Lipid A-modified Escherichia coli can produce porcine parvovirus virus-like particles with high immunogenicity and minimal endotoxin activity.

BACKGROUND: A cost-effective Escherichia coli expression system has gained popularity for producing virus-like particle (VLP) vaccines. However, the challenge lies in balancing the endotoxin residue and removal costs, as residual endotoxins can cause inflammatory reactions in the body. RESULTS: In this study, porcine parvovirus virus-like particles (PPV-VLPs) were successfully assembled from Decreased Endotoxic BL21 (BL21-DeE), and the effect of structural changes in the lipid A of BL21 on endotoxin activity, immunogenicity, and safety was investigated. The lipopolysaccharide purified from BL21-DeE produced lower IL-6 and TNF-α than that from wild-type BL21 (BL21-W) in both RAW264.7 cells and BALB/c mice. Additionally, mice immunized with PPV-VLP derived form BL21-DeE (BL21-DeE-VLP) showed significantly lower production of inflammatory factors and a smaller increase in body temperature within 3 h than those immunized with VLP from BL21-W (BL21-W-VLP) and endotoxin-removed VLP (ReE-VLP). Moreover, mice in the BL21-DeE-VLP immunized group had similar levels of serum antibodies as those in the BL21-W-VLP group but significantly higher levels than those in the ReE-VLP group. Furthermore, the liver, lungs, and kidneys showed no pathological damage compared with the BL21-W-VLP group. CONCLUSION: Overall, this study proposes a method for producing VLP with high immunogenicity and minimal endotoxin activity without chemical or physical endotoxin removal methods. This method could address the issue of endotoxin residues in the VLP and provide production benefits.

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.

Six underreported viral diseases of domesticated and wild swine in Africa: Implications and perspectives.

Pig production is increasing annually in Africa as it is recognized as a significant source of income, livelihood and food security, particularly in rural communities. Understanding the circulating swine pathogens is crucial for the success of this emerging industry. Although there is extensive data available on the African swine fever virus due to its devastating impact on pig production, knowledge about the presence of other viral swine pathogens on the continent is still extremely limited. This review discusses what is currently known about six swine pathogens in Africa: classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus-2, porcine circovirus-3, porcine parvovirus-1, and pseudorabies virus. Gaps in our knowledge are identified and topics of future focus discussed.

Proteomics analysis of PK-15 cells infected with porcine parvovirus and the effect of PCBP1 on PPV replication.

Porcine parvovirus (PPV) is one of the most important pathogens that cause reproductive failure in pigs. However, the pathogenesis of PPV infection remains unclear. Proteomics is a powerful tool to understand the interaction between virus and host cells. In the present study, we analyzed the proteomics of PPV-infected PK-15 cells. A total of 32 and 345 proteins were differentially expressed at the early and replication stages, respectively. Subsequent gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed these differentially expressed proteins were significantly enriched in pathways including toll-like receptor signaling pathway, tumor necrosis factor signaling pathway, and viral carcinogenesis. The expression of poly (rC) binding protein 1 (PCBP1) was observed to decrease after PPV infection. Overexpressed or silenced PCBP1 expression inhibited or promoted PPV infection. Our studies established a foundation for further exploration of the multiplication mechanism of PPV. IMPORTANCE: Porcine parvovirus (PPV) is a cause of reproductive failure in the swine industry. Our knowledge of PPV remains limited, and there is no effective treatment for PPV infection. Proteomics of PPV-infected PK-15 cells was conducted to identify differentially expressed proteins at 6 hours post-infection (hpi) and 36 hpi. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that various pathways participate in PPV infection. Poly (rC) binding protein 1 was confirmed to inhibit PPV replication, which provided potential targets for anti-PPV infection. Our findings improve the understanding of PPV infection and pave the way for future research in this area.

Chimeric Porcine Parvovirus VP2 Virus-like Particles with Epitopes of South African Serotype 2 Foot-and-Mouth Disease Virus Elicits Specific Humoral and Cellular Responses in Mice.

Southern Africa Territories 2 (SAT2) foot-and-mouth disease (FMD) has crossed long-standing regional boundaries in recent years and entered the Middle East. However, the existing vaccines offer poor cross-protection against the circulating strains in the field. Therefore, there is an urgent need for an alternative design approach for vaccines in anticipation of a pandemic of SAT2 Foot-and-mouth disease virus (FMDV). The porcine parvovirus (PPV) VP2 protein can embed exogenous epitopes into the four loops on its surface, assemble into virus-like particles (VLPs), and induce antibodies and cytokines to PPV and the exogenous epitope. In this study, chimeric porcine parvovirus VP2 VLPs (chimeric PPV-SAT2-VLPs) expressing the T-and/or B-cell epitopes of the structural protein VP1 of FMDV SAT2 were produced using the recombinant pFastBac™ Dual vector of baculoviruses in Sf9 and HF cells We used the Bac-to-Bac system to construct the recombinant baculoviruses. The VP2-VLP--SAT2 chimeras displayed chimeric T-cell epitope (amino acids 21-40 of VP1) and/or the B-cell epitope (amino acids 135-174) of SAT FMDV VP1 by substitution of the corresponding regions at the N terminus (amino acids 2-23) and/or loop 2 and/or loop 4 of the PPV VP2 protein, respectively. In mice, the chimeric PPV-SAT2-VLPs induced specific antibodies against PPV and the VP1 protein of SAT2 FMDV. The VP2-VLP-SAT2 chimeras induced specific antibodies to PPV and the VP1 protein specific epitopes of FMDV SAT2. In this study, as a proof-of-concept, successfully generated chimeric PPV-VP2 VLPs expressing epitopes of the structural protein VP1 of FMDV SAT2 that has a potential to prevent FMDV SAT2 and PPV infection in pigs.

Prevalence of viral agents causing swine reproductive failure in Korea and the development of multiplex real-time PCR and RT-PCR assays.

This study aimed to investigate the prevalence of viral agents causing reproductive failure in pigs in Korea. In addition, two types of multiplex real-time PCR (mqPCR) were developed for the simultaneous detection of Aujeszky's disease virus (ADV) and porcine parvovirus (PPV) in mqPCR and encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV) in reverse transcription mqPCR (mRT-qPCR). A total of 150 aborted fetus samples collected from 2020 to 2022 were analyzed. Porcine reproductive and respiratory syndrome virus was the most prevalent (49/150 32.7%), followed by porcine circovirus type 2 (31/150, 20.7%), and PPV1 (7/150, 4.7%), whereas ADV, EMCV, and JEV were not detected. The newly developed mqPCR and mRT-qPCR could simultaneously detect and differentiate with high sensitivities and specificities. When applied to aborted fetuses, the newly developed mqPCR for PPV was 33.3% more sensitivities than the previously established diagnostic method. Amino acid analysis of the VP2 sequences of PPV isolates revealed considerable similarity to the highly pathogenic Kresse strain. This study successfully evaluated the prevalence of viral agents causing reproductive failure among swine in Korea, the developed mqPCR and mRT-qPCR methods could be utilized as effective and accurate diagnostic methods for the epidemiological surveillance of ADV, PPV, EMCV, and JEV.

Molecular Survey on Porcine Parvoviruses (PPV1-7) and Their Association with Major Pathogens in Reproductive Failure Outbreaks in Northern Italy.

Successful reproductive performance is key to farm competitiveness in the global marketplace. Porcine parvovirus 1 (PPV1) has been identified as a major cause of reproductive failure, and since 2001 new species of porcine parvoviruses, namely PPV2-7, have been identified, although their role is not yet fully understood yet. The present study aimed to investigate PPVs' presence in reproductive failure outbreaks occurring in 124 farms of northern Italy. Fetuses were collected from 338 sows between 2019 and 2021 and tested for PPVs by real-time PCR-based assays and for other viruses responsible for reproductive disease. At least one PPV species was detected in 59.7% (74/124) of the tested farms. In order, PPV1, PPV5, PPV6, PPV7 and PPV4 were the most frequently detected species, whereas fewer detections were registered for PPV2 and PPV3. Overall, the new PPV2-7 species were detected in 26.6% (90/338) of the cases, both alone or in co-infections: PCV-2 (7.1%, 24/338), PCV-3 (8.2%, 28/338), and PRRSV-1 (6.2%, 21/338) were frequently identified in association with PPVs. Single PPVs detections or co-infections with other agents commonly responsible for reproductive failure should encourage future studies investigating their biological, clinical, and epidemiological role, for a better preparedness for potential emerging challenges in intensive pig production.

Identification and in vitro characterization of a novel porcine parvovirus 6 in Russia.

Porcine parvovirus 6 (PPV6) was first identified in aborted swine fetuses in China in 2014. Since its identification, an increased number of PPV6 cases have been reported in many countries with developed pig breeding. In this study, the first identification of porcine parvovirus 6 in Russia, its phylogenetic analysis, and its characterization in vitro are reported. During the investigation, 521 serum samples collected from pigs of different ages from seven regions of the Russian Federation were tested. In four regions, the DNA of the virus was detected. The overall prevalence of porcine parvovirus 6 in Russia was 9.4%. Fattening pigs were the group with the most frequent detection of the virus genome. Phylogenetic analysis of the Russian isolate detected in a domestic boar indicated high homology with strains from Spain. In vitro studies revealed that the most promising cell cultures for PPV6 isolation are SPEV and SK. Our results demonstrated that PPV6 induced typical apoptotic features in cells, including DNA fragmentation, chromatin margination, nuclear condensation, pyknosis of nuclei, symplast formation, and various pathological mitoses.

Isolation, characterization, and phylogenetic analysis of two new porcine parvovirus 1 isolates from Northern China.

Porcine parvovirus (PPV) is a pathogen of infectious reproductive disease, which can cause stillbirth, mummification, embryo death, and infertility (SMEDI) syndrome in pigs. The objective of this study was to gain new insights into the evolution and phylogeny of the PPV1 genome. In this study, we isolated two new PPV1 (HLJ202108-Y and SDLC202109) from northern China and sequenced their whole genomes. The new isolates were found to have three amino acid substitutions (K195R, K562R, and S578P) in nonstructural protein 1. The VP2 amino acid site contained nine nonsynonymous substitutions, including six substitutions of the Kresse strain corresponding to the NADL-2 strain and three substitutions of A414S, S436T, and N555K. Genetic evolution analysis was conducted on 107 reference sequences available in the GenBank database, and 4-5 PPV1 taxa were defined. The new isolates were in the same phylogenetic cluster as strain 27a. The changes in the cluster, specifically marker amino acids, and their potential role in enhancing pathogenicity are discussed in this study. Furthermore, the evolutionary tree map results showed that the strains in China were evolving in two directions: one was becoming increasingly similar to early NADL-2 strains, while the other was evolving toward 27a-like strains. We also compared the proliferation ability of the isolated strains in susceptible cells by analyzing the multistep growth curves. The results showed that the virulence titer of the mutant strain was high. In summary, this study introduced the latest changes in PPV and discussed the virus characteristics that were considered to affect virulence.

Detection and genetic evolution analysis of porcine parvovirus type 7 (PPV7) in Fujian Province.

Porcine parvovirus (PPV) is an important pathogen causing reproductive disorders in sows, with clinical symptoms including stillbirth, mummified fetuses, embryonic dysplasia and death, and sow infertility. Porcine parvovirus 7 (PPV7) is a recently discovered type of PPV and its widespread distribution and rapid evolution has caused huge economic losses in the pig industry. To investigate the molecular epidemiology of PPV7 in Fujian Province, China, we collected 491 blood samples and 72 tissue samples from diseased pigs in large-scale pig farms across selected areas of Fujian Province from 2019 to 2022. PPV7 infection was determined using real-time quantitative PCR, and positive samples underwent whole-genome amplification, sequencing, and subsequent homology, phylogenetic, and recombination analyses. The PPV7 positive detection rate was 25.73% (145/563) in Fujian Province, among which the positive rate of blood and tissue samples was 26.47% (130/491) and 20.83% (15/72), respectively. The nucleotide sequence homology among the 29 PPV7 whole-genome sequences obtained in this study was 90.0%-97.2%, whereas that with 128 reference strains from China and other countries was 88.9%-98.1%. Six strains had partial nucleotide deletions or insertions. Phylogenetic analysis based on the whole-genome sequences classified the 29 PPV7 strains and 128 reference strains into eight subtypes (PPV7a-PPV7h), and PPV7h was the predominant subtype in Fujian Province. Recombination analysis revealed evidence of inferred recombination events in the genomes of four strains. This study provides significant insights into the molecular characteristics of PPV7 in Fujian Province and serves as a crucial foundation for further advancements in PPV7 prevention and control strategies.

Detection of porcine circovirus 2, porcine parvovirus 1, and torque teno sus virus k2a in wild boars from northeastern Patagonia, Argentina.

Wild boars can act as a reservoir of pathogenic viruses that affect the pig industry. Here, we assessed the presence of porcine circovirus 2, porcine parvovirus 1, and torque teno sus virus k2a in wild boars in northeastern Patagonia (Argentina). Total DNA was extracted from the tonsils of 27 animals (collected between early 2016 and mid-2019) and used to prepare sample pools, which were subjected to viral detection through two-round PCR assays. Sequencing of the amplification products and phylogenetic analysis confirmed the occurrence of all of the aforementioned infectious agents.

A Phylogeographic Analysis of Porcine Parvovirus 1 in Africa.

Porcine parvovirus 1 (PPV1) is recognized as a major cause of reproductive failure in pigs, leading to several clinical outcomes globally known as SMEDI. Despite being known since the late 1960s its circulation is still of relevance to swine producers. Additionally, the emergence of variants such as the virulent 27a strain, for which lower protection induced by vaccines has been demonstrated, is of increasing concern. Even though constant monitoring of PPV1 using molecular epidemiological approaches is of pivotal importance, viral sequence data are scarce especially in low-income countries. To fill this gap, a collection of 71 partial VP2 sequences originating from eight African countries (Burkina Faso, Côte d'Ivoire, Kenya, Mozambique, Namibia, Nigeria, Senegal, and Tanzania) during the period 2011-2021 were analyzed within the context of global PPV1 variability. The observed pattern largely reflected what has been observed in high-income regions, i.e., 27a-like strains were more frequently detected than less virulent NADL-8-like strains. A phylogeographic analysis supported this observation, highlighting that the African scenario has been largely shaped by multiple PPV1 importation events from other continents, especially Europe and Asia. The existence of such an international movement coupled with the circulation of potential vaccine-escape variants requires the careful evaluation of the control strategies to prevent new strain introduction and persistence.

Evidence indicating transmission of porcine parvovirus 1 between warthogs and domestic pigs in Namibia.

Thirty swine samples collected from different regions of Namibia between 2019 and 2020 were screened for the presence of porcine parvovirus 1 (PPV1) by PCR. Eleven samples (37%) were positive. Phylogenetic analysis of a partial sequence of the structural protein gene (VP2) identified two distinct clusters, one which contained sequences that were highly similar to PPV1 previously identified in warthogs in Namibia. These results indicate possible PPV1 transmission between warthogs and domestic pigs and highlight the importance of wildlife as sources of pathogens.

Development of multiplex PCR assay for simultaneous detection of African swine fever, porcine circo and porcine parvo viral infection from clinical samples.

A diagnostic method for simultaneously detecting and distinguishing African Swine Fever (ASF), porcine circovirus type 2 (PCV2), and porcine parvovirus (PPV) in clinical specimens is critical for differential diagnosis, monitoring, and control in the field. Three primer pairs were designed and used to create a multiplex PCR assay. In addition, 356 porcine post mortem tissue samples from various parts of India's North Eastern region were tested by the developed multiplex PCR assay to demonstrate its accuracy. Using the designed primers, each of the ASF, PCV2 and PPV target genes was amplified, but no other porcine virus genes were detected. The assay's limit of detection was 102 copies/µl of PCV2, PPV, or ASFV. The detection of PCV2, PPV, and ASF in postmortem tissue samples revealed that they are co-circulating in India's North-Eastern region. The percentage positivity (PP) for PCV2, PPV and ASF single infection were 7.02% (25/356), 3.93% (14/356), and 3.37% (12/356), respectively, while the PP for PCV2& PPV co-infection was 2.80% (10/356), ASF & PCV2 co infection was 1.4% (5/356) and the ASF, PPV& PCV2 co-infection was1.40% (5/356). The results also indicate that the ASF can infect pigs alongside PCV and PPV.

Coinfections of African swine fever virus, porcine circovirus 2 and 3, and porcine parvovirus 1 in swine in Nigeria.

As pig production increases in Africa, it is essential to identify the pathogens that are circulating in the swine population to assess pig welfare and implement targeted control measures. For this reason, DNA samples collected from pigs in Nigeria in the context of African swine fever monitoring were further screened by PCR for porcine circovirus 2 (PCV-2), porcine circovirus 3 (PCV-3), and porcine parvovirus 1 (PPV1). Forty-seven (45%) pigs were positive for two or more pathogens. Sequence analysis identified PCV-2 genotypes a, b, and d, while limited genetic heterogenicity was observed among PCV-3 strains. All except one of the PPV1 sequences were genetically distinct from those previously identified in other countries.

Chaperonin CCT5 binding with porcine parvovirus NS1 promotes the interaction of NS1 and COPƐ to facilitate viral replication.

Porcine parvovirus (PPV) is an important pathogen causing reproductive disorders in first pregnant sows. The non-structure protein NS1 of PPV is a multifunctional protein playing a key role in viral replication. Chaperonin-containing T-complex polypeptide complex (CCT), containing CCT1-CCT8 subunits, belongs to the type II chaperones that interact with proteins to help in folding and maintaining. In this study, CCT5, for the first time, was found to be one of the host interacting proteins of PPV NS1, and CCT5 was directly bound with NS1. Interference of CCT5 expression by specific siRNA and knockout of CCT5 expression by CRISPR/Cas9 suppressed PPV replication, while overexpression of CCT5 promoted PPV replication in PK-15 cells. The interaction of CCT5 and PPV NS1 was dependent on the 36-42 aa motif at the N-terminal end of NS1. More importantly, CCT5 was also found interacting with COPƐ, which has previously been demonstrated to promote PPV replication by regulating type I interferon. Interference and knockout of CCT5 expression significantly reduced the interaction of PPV NS1 and host protein COPƐ, and promoted the IFN-ß expression. These results show that CCT5 mediates the interaction of PPV NS1 and COPƐ to regulate viral replication, providing new insight into the mechanism of PPV replication.

Allele-Specific Dual PCRs to Identify Members of the 27a Cluster of PPV.

Porcine Parvovirus (PPV) is one of the most important infectious agents causing severe reproductive failure in pigs. In the last two decades a particular, a novel genotype emerged in Europe and PPV-27a was named as the prototype of this genetic cluster. It was suggested that members of the PPV-27a cluster may adversely influence effective vaccination against PPV. For a reliable updated 27a definition, we aligned 93 databank-deposited partial or full nucleotide and protein sequences of the VP2 of different PPV isolates. We confirmed that the 27a cluster could indeed be distinguished from other members of the species, however, some divergences were identified compared to earlier defined genetic markers. Based on genetic differences, we developed a dual allele-specific polymerase chain reaction for the easy and quick discrimination of members of the 27a cluster from other PPV strains. The detection limit of dual PCR was found <1.66 × 104 copies/reaction. To sensitize and make it more user friendly, the method was further developed for qPCR application with fluorescent probes. Regarding the detection limit of the two PCRs (<1.66 × 104 copies/reaction of the dual PCR versus <2.40 × 102 copy/reaction of the dual qPCR), approximately two log improvement was achieved in the sensitivity of the method.

Molecular detection of porcine parvovirus 1-associated reproductive failure in southern India.

This study used 56 aborted and stillborn fetuses from organized swine farms in Tamil Nadu and Kerala, southern states of India. All samples were screened by using a PCR assay that targets the NS1 gene for PPV. Furthermore, the PCR positive samples were subjected to amplification of the VP2 gene of PPV1 with designed primers and sequenced for further study. The PCR screening of 56 samples found that 14.3% (n = 8) were positive for PPV genome. According to VP2 gene-based PCR for PPV1, 897 bp specific amplicons were detected in all eight of the samples. Two of the eight positive samples (L17 and T5) were sequenced and annotated randomly. The BLAST analysis of contig sequence INDTNCHN-T5 revealed 100% sequence homology with Chinese PPV1genome, whereas sequence from INDTNCHN-L17 revealed 99.43% sequence homology with Spain, Chinese, and German. PPV1 sequences and both the sequences INDTNCHN-T5 and INDTNCHN-L17 were submitted to the GenBank under the accession numbers MW822566 and MW822567 respectively. A phylogenetic analysis of the sequences in this study revealed specific grouping along with PPV1 strains in cluster E. Amino acid analysis of both isolated sequences in addition to the reference sequence from PPV1 showed variations in position 215 (I to T) in both the isolates, variation at position 228 (Q to E) in T5 isolate and variations at position 59 (L to M) and 314 (K to E) in L17 isolate. This study represents the first report of PPV1 cluster E in Tamil Nadu, southern India.

Rapid and Visual Detection of Porcine Parvovirus Using an ERA-CRISPR/Cas12a System Combined With Lateral Flow Dipstick Assay.

Porcine parvovirus (PPV) is one of the important causes of pig reproductive diseases. The most prevalent methods for PPV authentication are the polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, and quantitative real-time PCR. However, these procedures have downsides, such as the fact that they take a long time and require expensive equipment. As a result, a rapid, visible, and economical clinical diagnostic strategy to detect PPV is necessary. In this study, three pairs of crRNA primers were designed to recognize the VP2 gene, and an ERA-CRISPR/Cas12a system for PPV detection was successfully developed. The approach involved isothermal detection at 37°C, and the method can be used for visual inspection. The detection limit of the ERA-CRISPR/Cas12a system was 3.75 × 102 copies/µL, and no cross reactions with other porcine viruses were found. In view of the preceding, a rapid, visible, and low-cost nucleic acid testing approach for PPV has been developed using the ERA-CRISPR/Cas12a system.

Development of a novel one-step triplex PCR assay for the simultaneous detection of porcine circovirus type 2, porcine parvovirus and classical swine fever virus in a single tube.

Co-infection of multiple pathogens complicates diagnosis, treatment and preventive measures based on clinical signs. Therefore, reliable diagnostic tool for timely reporting of suspected diseases is very much essential. A novel one-step triplex PCR assay was developed and evaluated for simultaneous detection of three important viruses namely porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and classical swine fever virus (CSFV) involved in reproductive problems in pigs. Each of the three pairs of oligonucleotide primers exclusively amplified the targeted fragment of the specific viruses. The multiplex PCR assay was found to be sensitive in detecting at least 300 pg of viral genomic DNA or RNA from a mixture of three viruses in a reaction. No amplification was obtained from other common viruses or pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), porcine group A rotavirus (PoRVA), Escherichia coli and Staphylococcus aureus thereby indicating that the developed multiplex PCR has high specificity. Because of the sensitivity and specificity, the developed multiplex PCR assay will be a useful tool for clinical diagnosis of mixed infections of DNA and RNA viruses in pigs.