Porcine Circovirus Type Research Articles

Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method.

Simple SummaryPorcine epidemic diarrhea (PED) is a severe disease which has led to tremendous economic losses in the swine industry all over the world. The early detection of its pathogen (PEDV) is vital to prevent and cure this disease. Here, we report the development of a new visual diagnostic test for PEDV—the reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. This new assay proved to be specific and sensitive when applied to clinical specimens.Porcine epidemic diarrhea virus (PEDV) can cause severe infectious porcine epidemic diarrhea (PED) and infect different ages of pigs, resulting in sickness and death among suckling pigs. For PEDV detection, finding an effective and rapid method is a priority. In this study, we established an effective reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for PEDV detection. Three sets of primers, specific for eight different sequences of the PEDV N gene, were designed in this study. The optimized RT-LAMP amplification program was as follows: 59 min at 61.9 °C and 3 min at 80 °C. The RT-LAMP results were confirmed with the addition of SYBR Green I fluorescence dye and with the detection of a ladder-like band by conventional gel electrophoresis analysis, which demonstrated a significant agreement between the two methods. The LOD of PEDV by RT-LAMP was 0.0001 ng/μL. Compared with RT-LAMP, the traditional RT-PCR method is 100-fold less sensitive. The RT-LAMP results had no cross-reaction with porcine parvovirus (PPV), porcine circovirus type 1 (PCV1), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), rotavirus (RV), transmissible gastroenteritis virus (TGEV) and porcine reproductive and respiratory syndrome virus (PRRSV). Consequently, the newly developed RT-LAMP method could provide an accurate and reliable tool for PEDV diagnosis.

Experimental infection of high health pigs with porcine circovirus type 2 (PCV2) and Lawsonia intracellularis.

BackgroundPorcine circovirus type 2 (PCV2) and Lawsonia intracellularis infections can cause enteritis in pigs. A Danish study showed a significantly higher probability of detecting PCV2 without concurrent L. intracellularis infection, indicating that one of these pathogens has an impact on the dynamics of the other. Therefore, a delayed co-infection model was set up, initially aiming at investigating the interaction between PCV2 and L. intracellularis in pigs challenged with PCV2 and 2 weeks later with L. intracellularis. But due to PCV2 contamination of the L. intracellularis inoculum the aim was revisited to describing the infection dynamics and pathogenesis of pigs infected with PCV2 followed by delayed simultaneous exposure to PCV2 and L. intracellularis. Twenty-four high-health piglets were divided into three groups of eight pigs (A, B, C) and inoculated at experimental day (EXD) 0 with mock (groups A and B) or PCV2 (group C), and at EXD 14 with mock (group A) or L. intracellularis/PCV2 (groups B and C). The pigs underwent daily clinical examination, and were necropsied at EXD 51–52. Furthermore, histology, immunohistochemistry, serology and PCR for PCV2 and L. intracellularis, and measurement of C-reactive protein were carried out.ResultsGroup A remained negative for PCV2 and L. intracellularis. Following inoculation with L. intracellularis/PCV2, no significant differences were observed between group B and C, however pigs already infected with PCV2 (group C) showed milder clinical signs and exhibited milder intestinal lesions, less shedding of L. intracellularis and developed higher L. intracellularis antibody titers than the pigs in group B that only received the combined infection. Though the differences between group B and C were non-significant, all results pointed in the same direction, indicating that the pigs in group B were more affected by the L. intracellularis infection compared to the pigs in group C.ConclusionsPrevious exposure to PCV2 had limited impact on the subsequent exposure to a combined L. intracellularis/PCV2 inoculation. However, there was a tendency that the infection dynamics of PCV2 and development of antibodies to PCV2 and L. intracellularis were altered in pigs previously exposed to PCV2. These differences should be confirmed in further experimental trials.

Molecular Epidemiology and Genetic Characterization of PCV2 Circulating in Wild Boars in Southwestern Ethiopia.

Porcine circovirus type 2 (PCV2) is one of the most relevant infectious agents affecting domestic pigs. Recently, a surprising PCV2 genetic heterogenicity has been reported in Africa. Nevertheless, the knowledge of the epidemiology of PCV2 in African countries, in both domestic and wild species, is limited and sparse. Having this in mind, in the present study, the PCV2 circulation and its molecular epidemiology in Southwestern Ethiopia have been investigated by collecting 64 samples from domestic pigs, wild boars, and warthogs. PCV2 genome presence was detected and quantified using qPCR and ORF2 sequencing was attempted on positive samples. Ten samples, 8 wild boars, 1 domestic pig, and 1 warthog, tested PCV2 positive. Complete ORF2 sequences were obtained from 5 wild boars; 4 of those were classified as PCV2d and 1 as PCV2b. Both PCV2b and PCV2d were related to strains of Asian origin, most commonly from China. The role of this country in the exportation of PCV2 strains in Ethiopia, and Africa in general, might be supported by the crescent economic relationship between the two continents. The obtained evidence also testifies to the inadequacy and/or poor application of biosecurity measures separating wild and domestic animals. Further, extensive and systematic studies should be performed to more deeply characterize the molecular epidemiology of PCV2 in this region, in order to improve our understanding of these ecological niches in the evolution and dispersal of PCV2.

Detection of Porcine Circovirus Type 2a and Pasteurella multocida Capsular Serotype D in Growing Pigs Suffering from Respiratory Disease.

Simple SummaryBacterial and viral respiratory diseases are a major health problem in pig farms. Most often multiple infectious agents are involved in the development of clinical cases of respiratory diseases. It is necessary to conduct laboratory etiological tests on suspected cases. In January 2021, a 70-day-old pig herd in a pig farm in Hebei Province, China, had been suffering from respiratory problems. On the basis of this preliminary clinical diagnosis, this study carried out comprehensive laboratory detection including nucleic acid detection and sequencing, the isolation and identification of the pathogen, genetic evolution analysis, and genotype or serotype identification, as well as an analysis of the bacterial morphology characteristics and pathogenicity. The results showed that the co-infection of PCV2a and P. multocida capsular serotypes D was likely responsible for the disease. This study could provide important etiological information for effective control and treatment of the disease in pig farms.In order to diagnose a respiratory disease in a pig farm, the lungs, spleen, and lymph nodes of three dead pigs were collected for pathogen detection by PCR and isolation on the basis of preliminary clinical diagnosis. The virus isolate was identified by gene sequence analysis and Immunoperoxidase monolayer assay (IPMA). The bacterial isolate was identified by biochemical tests, 16S rDNA sequence analysis, and species- and serotype-specific PCR, and the pathogenicity was analyzed. Porcine circovirus type 2a (PCV2a) genotype from the lungs, spleen, and lymph nodes and Pasteurella (P.) multocida capsular serotypes D from the lungs were found. The PCV2a isolates could specifically bound the anti-PCV2-Cap polyclonal antibody. The 16S rDNA sequence of P. multocida isolates had 99.9% identity with that of the strain from cattle, and the isolate was highly pathogenic to mice. The results showed that the co-infection of PCV2a and P. Multocida capsular serotypes D should be responsible for the disease. The uncommon PCV2a is still prevalent in some pig farms besides the dominant PCV2d genotype. This study could provide important etiological information for effective control and treatment of the disease in pig farms.

A duplex fluorescent quantitative PCR assay to distinguish the genotype I and II strains of African swine fever virus in Chinese epidemic strains.

African swine fever (ASF) is a highly contagious hemorrhagic disease that affects domestic and wild pigs. A recent study reported that both ASF virus (ASFV) genotypes I and II have invaded farm-raised pigs in China, causing chronic infection and morbidity. To develop a duplex fluorescent quantitative PCR method to distinguish the ASFV genotypes I and II in Chinese epidemic strains, the probes and primers were designed based on the B646L sequences of genotypes I and II listed in the GenBank database. After optimizing the system, a duplex fluorescent quantitative PCR method for simultaneous detection of ASFV genotypes I and II B646L genes was successfully established. This method had no cross-reaction with Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), or Porcine Parvovirus (PPV), indicating that it has strong specificity. The sensitivity results indicated that the minimum detection limit of ASFV genotypes I and II B646L was 10 copies/Rxn. The inter- and intra-group coefficients of variation were both <3%, indicating that the method was highly reproducible. Therefore, the established duplex fluorescent quantitative PCR assay is important for the differential detection and epidemiological investigation of ASFV.

Survey of porcine circovirus type 2 and parvovirus in swine breeding herds of Colombia.

One of the consequences of the presentation of reproductive failures in sows is the economic losses in production because it alters the estimated values of the volume of production, decreasing the productivity of the farm. Porcine circovirosis by porcine circovirus 2 (PCV2) has been associated with reproductive disorders, and porcine parvovirus (PVP) is one of the pathological agents most related to the presentation of reproductive failure in pigs. In Colombia, there are reports of the presence of PCV2 through molecular techniques, and PVP through serum tests; however, in the department of Tolima, the prevalence of these two viruses is unknown. In this study, the aim was to establish a report of the prevalence of viruses in five municipalities of the department of Tolima-Colombia. Blood samples from 150 breeding sows of five municipalities in Tolima, Colombia, were obtained. Quantitative polymerase chain reaction (qPCR) was used to detect the PCV2 and PVP virus in the blood samples followed by PCR and sequencing of 16 PCR products of the amplification of the cap gene of PCV2. A phylogenetic tree was constructed to identify the genotype of the PCV2 virus. The presence of PCV2d in sows was detected in 135 samples (90%), as well as the identification of PVP in 2.6% of the samples. In addition, the phylogenetic analysis showed that 16 isolates were the PCV2d2 genotype. PCV2d and PVP were found to coinfect the females, and the identification of variability in regions in the predicted amino acid sequence of the PCV2 capsid may be associated with virus pathogenicity.

The Decoy Epitopes Elicit Swine Antibodies by Connecting to the Carboxyl-terminal Epitope of the Capsid Protein of Porcine Circovirus Type 2

The Decoy Epitopes Elicit Swine Antibodies by Connecting to the Carboxyl-terminal Epitope of the Capsid Protein of Porcine Circovirus Type 2

Surface Display of porcine circovirus type 2 antigen protein cap on the spores of bacillus subtilis 168: An effective mucosal vaccine candidate.

The oral mucosal vaccine has great potential in preventing a series of diseases caused by porcine circovirus type 2 (PCV2) infection. This study constructed a recombinant Bacillus subtilis RB with PCV2 Capsid protein (Cap) on its spore surface and cotB as a fusion partner. The immune properties of the recombinant strain were evaluated in a mouse model. IgA in intestinal contents and IgG in serum were detected by enzyme-linked immunosorbent assay (ELISA). The results demonstrated that recombinant spores could activate strong specific mucosal and humoral immune responses. In addition, spores showed good mucosal immune adjuvant function, promoting the proliferation of CD3+, CD4+ and CD8+ T cells and other immune cells. We also found that the relative expression of inflammatory cytokines such as IL-1β, IL-6, IL-10, TNF-α and IFN in the small intestinal mucosa was significantly up-regulated under the stimulation of recombinant bacteriophage. These effects are important for the balance of Th1/Th2-like responses. In summary, our results suggest that recombinant B. subtilis RB as a feed additive provides a new strategy for the development of novel and safe PCV2 mucosal subunit vaccines.

PCV3-associated reproductive failure in pig herds in Brazil.

Porcine circovirus type 3 (PCV3) has been widely detected worldwide in healthy and sick pigs. Recently its association with clinical disease and reproductive failure has been proven through the detection of intralesional viral mRNA in affected pigs. This study aims to describe the occurrence of PCV3-associated reproductive failure (abortions) in sow herds in southern Brazil. Eleven fetuses from five different litters from two herds were analyzed. These herds reported an increase in the rate of late-gestation abortions, stillbirths, and the percentage of mummified piglets. At gross examination, six of the fetuses had large caudally rotated ears and one fetus was mummified. Microscopically, multisystemic vasculitis, lymphocytic interstitial pneumonia, myocarditis, and encephalitis were observed. These six fetuses with gross and histological lesions were positive in qPCR analysis for PCV3, and PCV3 transcription was shown through in situ hybridization (ISH-RNA) within the histologic lesions. Samples from all 11 fetuses tested negative in PCR exam for Porcine Circovirus type 1 and 2, Porcine Reproductive and Respiratory Syndrome, Porcine Parvovirus, and Atypical Porcine Pestivirus. Furthermore, based on the ORF2 analysis, the PCV3a clade was identified. This is the first report of PCV3a-associated reproductive failure in pig herds in South America.

Porcine Circovirus Type 2 Vaccines: Commercial Application and Research Advances.

Porcine circovirus type 2 (PCV2) infection can lead to porcine circovirus-associated disease (PCVAD), causing great economic losses to the global swine industry. Conventional vaccination programs are a major measure in the prevention and control of this disease. Currently, there are 5 commercially available PCV2 vaccines in the international market and 10 kinds commercially available PCV2 vaccines in the Chinese market that confer good efficacy against this virus by alleviating clinicopathological manifestations and enhancing growth performance in pigs. In addition, diverse experimental PCV2 vaccines with protective efficiency have been developed, including attenuated chimeric, nucleic acid, subunit, multivalent, and viral-vectored vaccines. These experimental vaccines have been shown to be relatively effective in improving the efficiency of pig production and simplifying prevention procedures. Adjuvants can be used to promote vaccines with higher protective immunity. Herein, we review the application of multiple commercial vaccines over the years and research advances in experimental vaccines, which provide the possibility for the development of superior vaccines to successfully prevent and control PCV2 infection in the future.

Pathological Evaluation of Porcine Circovirus 2d (PCV2d) Strain and Comparative Evaluation of PCV2d and PCV2b Inactivated Vaccines against PCV2d Infection in a Specific Pathogen-Free (SPF) Yucatan Miniature Pig Model.

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen that causes porcine circovirus-associated diseases (PCVADs). The objective of this study was to evaluate the use of specific pathogen-free Yucatan miniature pigs (YMPs) as an experimental model for PCV2d challenge and vaccine assessment because PCV2-negative pigs are extremely rare in conventional swine herds in Korea. In the first experiment, every three pigs were subjected to PCV2d field isolate or mock challenge. During three weeks of experiments, the PCV2d infection group exhibited clinical outcomes of PCVAD with high viral loads, lymphoid depletion, and detection of PCV2d antigens in lymphoid organs by immunohistochemistry. In the second experiment, three groups of pigs were challenged with PCV2d after immunization for three weeks: a nonvaccinated group (three pigs), a PCV2b-Vac group vaccinated with a commercial PCV2b-based inactivated vaccine SuiShot® Circo-ONE (five pigs), and a PCV2d-Vac group vaccinated with an experimental PCV2d-based inactivated vaccine (five pigs). During the three weeks of the challenge period, nonvaccinated pigs showed similar clinical outcomes to those observed in the PCV2d infection group from the first experiment. In contrast, both the PCV2b and PCV2d vaccinations produced good levels of protection against PCV2d challenge, as evidenced by reduced viral loads, improved growth performance, high virus-neutralizing antibody titers, and less development of PCV2-associated pathological lesions. Taken together, these data suggest that YMPs could be an alternative model for PCV2 challenge experiments, and these animals displayed typical clinical and pathological features and characteristics of protective immunity induced by the vaccines that were consistent with those resulting from PCV2 infections in conventional pigs.

An ethyl acetate fraction of flavonoids from Polygonum hydropiper L. exhibits an anti-inflammatory activity in PCV2-infected porcine alveolar macrophages via PI3K/Akt and NF-κB pathways.

Porcine circovirus type 2 (PCV2) widelyexists in swine production systems causing porcine circovirus diseases (PCVD) which is associated with significant economic losses. Polygonum hydropiper L. was used as a traditional Chinese medicine to treat a variety of diseases. This study was carried out to investigate anti-inflammatory activity of the ethyl acetate fraction of flavonoids from Polygonum hydropiper L. (FEA) in PCV2-induced porcine alveolar macrophages (3D4/2 cell line). The production of oxygen species (ROS) and the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-8 (IL-8) were detected to evaluate the anti-inflammatory activities of FEA. The translocation of nuclear factor-kappa B (NF-κB) and the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathways were investigated to document the potential anti-inflammatory mechanisms. In PCV2 induced 3D4/2 cells, FEA treatment significantly reduced the production of ROS, and sharply down-regulated the levels of TNF-α, IL-1β and IL-8 in both secretion and mRNA expression level. The FEA also decreased the mRNA expression of Akt and NF-κB p65, reduced the transfer of p65 to nuclear, and inhibited the activation ofPI3K/Akt signaling pathway. The findings suggest that FEA exhibited an anti-inflammatory activity in vitro and could be used as a candidate in treatment of inflammation induced by PCV2 infection.

A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus, African swine fever virus, and atypical porcine pestivirus

With the implementation of the C-strain vaccine, classical swine fever (CSF) has been under control in China, which is currently in a chronic atypical epidemic situation. African swine fever (ASF) emerged in China in 2018 and spread quickly across the country. It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety. Atypical porcine pestivirus (APPV) was first detected in Guangdong Province, China, in 2016, which mainly harms piglets and has a local epidemic situation in southern China. These three diseases have similar clinical symptoms in pig herds, which cause considerable losses to the pig industry. They are difficult to be distinguished only by clinical diagnosis. Therefore, developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential. In this study, three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV (5′ UTR), African swine fever virus (ASFV) (B646L), and APPV (5′ UTR), followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay. The results showed that the method did not cross-react with other swine pathogens (porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), pseudorabies virus (PRV), porcine parvovirus (PPV), and bovine viral diarrhea virus BVDV). The sensitivity results showed that CSFV, ASFV, and APPV could be detected as low as 1 copy mL−1; the repeatability results showed that the intra-assay and inter-assay coefficient of variation of ASFV, CSFV, and APPV was less than 1%. Twenty-two virus samples were detected by the multiplex real-time PCR, compared with national standard diagnostic and patented method assay for CSF (GB/T 27540–2011), ASF (GB/T 18648–2020), and APPV (CN108611442A), respectively. The sensitivity of this triple real-time PCR for CSFV, ASFV, and APPV was almost the same, and the compliance results were the same (100%). A total of 451 clinical samples were detected, and the results showed that the positive rates of CSFV, ASFV, and APPV were 0.22% (1/451), 1.3% (6/451), and 0% (0/451), respectively. This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV, ASFV, and APPV.

Pathomorphology, immunohistochemical, and molecular detection of an atypical porcine dermatitis and nephropathy syndrome (PDNS) due to PCV-2d-2 in naturally affected grower pigs of India

Porcine circovirus type 2 (PCV-2) is of great economic significance to porcine industry worldwide. PCV-2 variants and genotypes, alternating world over, are the etiological agent of several clinical syndromes such as porcine dermatitis and nephropathy syndrome (PDNS), post-weaning multi-systemic wasting syndrome (PMWS) and others in pigs. This study is reporting an atypical manifestation of PDNS in twelve grower pigs, 3- to - 4.5 months age and either sex, died of the disease, with predominant lesions of nephropathy and no obvious clinical lesions in skin. Necropsy examination of pigs showed lesions of petechial -to- ecchymotic hemorrhages in the kidneys and in the right auricular musculature of the hearts. Microscopic lesions in H & E sections of the kidneys showed acute glomerulonephritis, interstitial nephritis, and vasculitis, but the skin morphology and architecture remained unaltered in contrast to the pathognomonic lesions of PDNS described in the literature. Other syndromic associations of PDNS in these cases included-perimyocarditis, interstitial pneumonia, depleted lymphoid tissues, tonsillitis, enteritis, and meningo-encephalitis. The lesional sites in duplicate paraffin tissue sections of kidneys, heart, lungs, spleen, lymph nodes, intestine, and brain demonstrated PCV-2 antigen in the cytoplasm of cells as highlighted by the intense immunolabeling on IHC staining. The PCV-2 positive organs reconfirmed by PCR, targeting ORF2 gene, which yielded 481bp size of products. The sequencing results of 481bp products on phylogenetic analysis showed 94% similarity with that of PCV-2 sequences in the database that grouped into PCV2d-2 genotype. The present report confirms, probably for the first time, the atypical PDNS cases due to PCV2d-2 genotype in naturally affected grower pigs of India.

Characterization of the antigens in inactivated porcine circovirus type 2 vaccines and virus-like particle vaccines by high-performance size-exclusion chromatography coupled with multi-angle laser light scattering

This paper aims to detect the antigens in porcine circovirus type 2 (PCV2) vaccines by high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS). With purified inactivated PCV2 and PCV2 virus-like particles (VLP) as references, two inactivated vaccines (a and b) and two VLP vaccines (c and d) for PCV2 from four manufacturers were analyzed by HPSEC-MALLS after demulsification. The antigen peaks in HPSEC-MALLS were identified by PCV2 antigen test strips, Western blotting and transmission electron microscope (TEM). The repeatability and linearity of the method were investigated. The results showed the virus antigens in the two inactivated vaccines were eluted at about 13.3 min in HPSEC. The molecular weight of these antigens was 2.61×106 (±4.34%) Da and 2.40×106 (±2.51%) Da, respectively, as calculated by MALLS. The antigen peaks of the two VLP vaccines also appeared at 13.3 min and the molecular weight was 2.09×106 (±2.94%) Da and 2.88×106 (±11.85%) Da, respectively, which was close to the theoretical molecular weight of PCV2. Moreover, an antigen peak of VLP vaccine c was observed at 11.4 min and the molecular weight was 4.37×106 (±0.42%) Da. The antigen was verified to be the dimer of VLP by TEM. Vaccine d and purified Cap VLP antigens were tested repeatedly, and the RSD of the peak area (n=3) was all < 1.5%, indicating that the method was repeatable. The purified VLP were diluted in serial and tested for linearity. The result suggested good linear relationship between the peak area of VLP or VLP aggregates and the protein concentration of the sample with R2 of 0.999 and 0.997, respectively. Thus, the method met the requirement for quantification and aggregate analysis. This method is accurate and efficient in in vitro quality evaluation and improvement of PCV2 vaccine.

Porcine circovirus type 2 induces apoptosis by exosomal miR-125a-5p targeting Bcl-2 in porcine lymphocytes

In order to investigate the apoptosis triggered by porcine circovirus type 2 (PCV2) in lymphocytes and the underlying mechanism, the levels of apoptosis and the expression levels of miRNA were examined by flow cytometry, Western blotting and real-time PCR (qPCR). The mimics or inhibitors of miR-125a-5p, an apoptosis-related miRNA, were transfected into PK-15 cells, and the apoptosis rate was examined upon overexpression or inhibition of mir-125a-5p. The target gene of mir-125a-5p was predicted by bioinformatics method, and the regulation of mir-125a-5p on the target gene was analyzed by luciferase reporter assay. The expressions of Bcl-2, Bax, cytochrome C and caspase-3 were detected by Western blotting. The results showed that exosomes secreted by PK-15 cells infected with PCV2 significantly increased the lymphocyte apoptosis rate, which was dose-dependent in certain concentration range. The expression of miR-125a-5p was dramatically increased. The apoptosis rate was increased significantly in the cells transfected with miR-125a-5p. It was predicted that there were binding sites of miR-125a-5p at Bcl-2 3'UTR by TargetScan. The luciferase activity of wild-type pmir-Bcl-2 3'UTR was inhibited significantly by miR-125a-5p mimics, but that of mutant pmir-Bcl-2 3'UTR was not changed. By Western blotting, Bcl-2 was reduced significantly, while Bax, cytochrome C and caspase-3 increased significantly, and the ratio of Bcl-2/Bax was significantly decreased. These results showed that PCV2 up-regulated the expression of miR-125a-5p through exosomes, then inhibited the expression of Bcl-2 at both mRNA and protein level, activated mitochondrial apoptosis pathway and induced apoptosis in lymphocytes.

Porcine circovirus type 2 induces CHOP-ERO1α-ROS-mediated apoptosis in PK-15 cells

Porcine circovirus type 2 (PCV2) infection induces endoplasmic reticulum (ER) stress and oxidative stress. These cellular responses could be connected with apoptosis. However, the mechanisms that link ER stress and oxidative stress in PCV2-induced apoptosis are poorly characterized. Here, we demonstrate that PCV2 infection increased expression of proapoptotic protein C/EBP homologous protein (CHOP) and ER oxidoreductase 1 alpha (ERO1α). Inhibition of CHOP by RNA silencing or inhibition of ERO1α by short hairpin RNA or EN460 repressed PCV2-induced reactive oxygen species (ROS) generation, cytosolic calcium level, and apoptotic rate in PK-15 cells. Overexpression of ERO1α enhanced PCV2-induced oxidative stress, caspase-3 cleavage, and apoptosis rate. Treatment of PCV2-infected cells with ROS scavenger N-acetyl-L-cysteine downregulated PCV2-induced ROS production, cytosolic calcium level, and apoptosis rate, but intriguingly decreased expression of CHOP and ERO1α. Thus, we propose that PCV2 induces apoptosis through ER Stress via CHOP-ERO1α-ROS signaling in host cells.

Precise Assembly of Multiple Antigens on Nanoparticles with Specially Designed Affinity Peptides.

Antigen proteins, assembled on nanoparticles, can be recognized by antigen-presenting cells effectively to enhance antigen immunogenicity. The ability to simultaneously display multiantigens on the same nanoparticle could have numerous applications but remained technical challenges. Here, we described a method for precise assembly of multiple antigens on nanoparticles with specially designed affinity peptides. First, we designed and screened affinity peptides with high affinity and specificity, which could respectively target the key amino acid residues of classical swine fever virus (CSFV) E2 protein or porcine circovirus type 2 capsid protein (PCV2 Cap) accurately. Then, we conjugated the antigen proteins to poly(lactic acid-glycolic acid) copolymer (PLGA) and Gram-positive enhancer matrix (GEM) nanoparticles through the peptides and perfectly assembled two kinds of multiantigen display nanoparticles with different particle sizes. Subsequently, the immunological properties of the assembled nanoparticles were tested. The results showed that the antigen display nanoparticles could promote the maturation, phagocytosis, and proinflammatory effects of antigen-presenting cells (APCs). Besides, compared with the antigen proteins, multiantigen display nanoparticles could induce much higher levels of antibodies and neutralizing antibodies in mice. This strategy may provide a technical support for the study of protein structure and the research and development of polyvalent vaccines.

First complete genomic sequence analysis of porcine circovirus type 4 (PCV4) in wild boars

Porcine circovirus type 4 (PCV4), a unique circovirus with a different classification from other existing circovirus, was discovered in domestic pigs in several provinces of China. In this study, in order to investigate the epidemiology and genetic diversity of PCV4 in wild boars (Sus scrofa), a total number of 138 wild boar samples were collected from five different areas in Jiangxi Province of China, between January 2020 and December 2020. Taqman based real-time PCR were used to test PCV4 as well as PCV1, PCV2, and PCV3. Among 138 samples, 30 samples (21.7%) were positive for PCV1, 31 samples (22.5%) were positive for PCV2, 8 samples (5.8%) were positive for PCV3 and 27 samples (19.6%) were positive for PCV4, respectively. Some of the samples were co-infected with multiple PCVs. In this study, we successfully sequenced the complete genome of two PCV4 strains, which shared 98.5–99.8% of their genomic nucleotide similarity with the other five PCV4 strains discovered in domestic pigs. Phylogenetic analysis showed that the two PCV4 strains derived from wild boars were located in a closed relative branch with other PCV4 strains derived from domestic pigs, but were distinguished from other circovirus. These results of this study not only expand our understanding of the prevalence of PCVs, especially PCV4, in wild boars in Jiangxi province of China, but also showed the molecular epidemiology of PCV4. Nevertheless, the impact of wild boars infected with PCV4 on intensive farmed pigs industry remains to be further explored.

Epidemiological Survey of Four Reproductive Disorder Associated Viruses of Sows in Hunan Province during 2019-2021.

Simple SummaryPorcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), and classical swine fever virus (CSFV) are major causative agents contributing to the reproductive disorders in sows in China. This is the first research investigating the epidemiology and molecular characteristics of these four viruses in sows with reproductive disorders in China. In this study, 407 aborted-fetus samples were obtained from 89 pig farms in Hunan province from 2019 to 2021 for molecular detection and phylogenetic analysis of PRRSV, PRV, PCV2, and CSFV. Collectively, various patterns of singular infections or co-infections were identified in pig herds; PRRSV and PCV2, especially, were frequently detected in the collected specimens. Further phylogenetic analysis revealed the complex genotypes of each virus prevalent in pig populations in Hunan province. In conclusion, this study provides the latest information on the epidemiology and genotype diversity of four viruses in sows with reproductive diseases in Hunan province of China, which would contribute to developing effective strategies for disease control.Porcine reproductive disorders have been considered as the major factors that threaten pig industries worldwide. In this study, 407 aborted-fetus samples were obtained from 89 pig farms in Hunan province, to investigate the prevalence of four viruses associated with porcine reproductive disease, including porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), and classical swine fever virus (CSFV). Meanwhile, the target gene sequences of representative PRRSV (ORF5), PCV2 (ORF2), CSFV (E2), and PRV (gE) strains were amplified, sequenced, and analyzed. The results showed that the positive rates of PRRSV, PCV2, PRV, and CSFV among the collected samples were 26.29% (107/407), 52.83% (215/407), 6.39% (26/407), and 12.29% (50/407), respectively. Moreover, co-infection with two and three pathogens were frequently identified, with PCV2/PRRSV, PRRSV/CSFV, PRRSV/PRV, PCV2/CSFV, PCV2/PRV, and PRRSV/PCV2/CSFV mix infection rates of 9.09%, 3.19%, 2.95%, 3.69%, 2.21%, and 0.49%, respectively. Moreover, ORF5-based phylogenetic analysis showed that 9, 4, and 24 of 37 PRRSV strains belonged to the PRRSV2 lineages 1, 5, and 8, respectively. ORF2-based phylogenetic analysis revealed that PCV2d and PCV2b were prevalent in Hunan province, with the proportions of 87.5% (21/24) and 12.5% (3/24), respectively. An E2-based phylogenetic tree showed that all 13 CSFV strains were clustered with 2.1 subgenotypes, these isolates were composed of 2.1b (10/13) and 2.1c (3/13) sub-subgenotypes. A gE-based phylogenetic tree showed that all six PRV strains belonged to the genotype II, which were genetically closer to variant PRV strains. Collectively, the present study provides the latest information on the epidemiology and genotype diversity of four viruses in sows with reproductive diseases in Hunan province, China, which would contribute to developing effective strategies for disease control.