Development of a RPA-CRISPR/Cas12a based rapid visual detection assay for Porcine Parvovirus 7.

Introduction: Porcine Parvovirus (PPV) is a significant pathogen in the pig industry, with eight genotypes, including PPV7, identified since its emergence in 2016. Co-infections with viruses such as Porcine Circovirus 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) pose serious risks to swine health. Thus, there is an urgent need for rapid, sensitive, and specific detection methods suitable for use in field settings or laboratories with limited resources. Methods: We developed a CRISPR/Cas12a-based assay combined with recombinase polymerase amplification (RPA) for the rapid detection of PPV7. Specific RPA primers and five CRISPR RNAs (crRNAs) were designed to target a highly conserved region within the NS1 gene of PPV7. Optimization of crRNA and single-stranded DNA (ssDNA) concentrations was performed to enhance the assay's performance. Results: CrRNA optimization identified crRNA-05 as the optimal candidate for Cas12a-based detection of PPV7, as all synthesized crRNAs demonstrated similar performance. The optimal crRNA concentration was determined to be 200 nM, yielding consistent results across tested concentrations. For ssDNA optimization, the strongest fluorescence signal was achieved with 500 nM of the FAM-BHQ ssDNA receptor. The assay showed a minimal detection limit of 100copies/µl for PPV7, confirmed through fluorescence and lateral flow detection methods. Specificity testing indicated that only PPV7 DNA samples returned positive results, confirming the assay's accuracy. In tests of 50 lung tissue samples from diseased pigs, the RPA-Cas12a assay identified 29 positive samples (58%), surpassing the 22 positive samples (44%) detected by conventional PCR. This highlights the RPA-Cas12a method's enhanced detection capability and its potential utility in clinical surveillance and management of PPV7 in swine populations. Discussion: The RPA-Cas12a assay effectively detects PPV7 in clinical samples, enhancing disease surveillance and control in pigs. Its adaptability to resource-limited settings significantly improves PPV7 management and prevention strategies, thereby supporting the overall health and development of the pig industry.

The first report of porcine parvovirus 8 (PPV8) on the American continent is associated with pigs in Colombia with porcine respiratory disease.

Seven novel porcine parvoviruses (PPV2 to PPV8) have been discovered in the last two decades. The last one reported was PPV8 in China in 2022, which was proposed to be a member of the genus Protoparvovirus. Here, we report the first detection of PPV8 outside China - in two provinces from Colombia. Six out of 146 (4.1%) pigs showing porcine respiratory disease (PRD) tested positive for PPV8. Sequencing and phylogenetic analysis of two Colombian PPV8 isolates (GenBank database accession numbers PP335559 and PP335560) showed them to be members of the genus Protoparvovirus. Furthermore, PPV8 was detected in coinfections with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), which are associated with PRD.

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.

Isolation and characterization of porcine parvovirus in Vietnam.

Background and Aim: No study has successfully isolated parvovirus in Vietnam. This study aimed to isolate and characterize parvovirus strains indigenous in Vietnam for vaccine development against porcine parvovirus (PPV). Materials and Methods: We collected serum and stillbirth samples from six provinces in Vietnam, and PPV-positive samples were identified using a polymerase chain reaction. Parvovirus isolation was attempted using the PK-15 cells maintained in a minimum essential medium supplemented with 5% fetal bovine serum and 1% antibiotics (Penicillin-streptomycin). The cells were incubated at 37°C with 5% CO2. Virulence experiments were conducted on white primiparous sows to evaluate the virulence of the PPV strain through hemagglutination inhibition (HI) titers and fetus lesions. Results: We analyzed 360 serum and 32 stillbirth (liver and lungs) samples, revealing that 32/392 (8.2% ) of them were PPV-positive, all belonging to PPV1. Thirty-two PPV-positive samples were successfully isolated, with 100% identity as VP2 sequences. The phylogenetic tree revealed a close relationship with the Kresse strain (isolated from Canada in 1996) and the PPV1-0225-L-SD strain (isolated from China in 2022). Two PPV isolates (VC5 from Dongnai and TX7 from Thanhhoa) that exhibited high 50% tissue culture infectious dose titers were selected for the virulence experiment. On day 21, after injection, the HI antibody titers ranged from 10log2 to 12log2. On day 90, 71%-80% of fetuses were mummified. Conclusion: This study showed that the PPV infection rate in Vietnam was 8.2%. Thirty-two isolates belonged to PPV1. Two PPV strains, VC5 and TX7, were determined to be highly virulent by the results of HI titers after injection into gilts. VC5 and TX7 were determined to be good candidates for further research on PPV vaccines.

First Report of Porcine Parvovirus 8 in Europe: Widespread Detection and Genetic Characterization on Commercial Pig Farms in Hungary and Slovakia.

Porcine parvovirus 8 (PPV8), a novel virus in the Parvoviridae family, was first identified in 2022 in lung samples of domestic pigs from China. Retrospective analyses showed that it had been circulating in China since 1998, but no other countries had reported its presence so far. A recent study conducted in South America did not detect any PPV8-positive samples in that region. Here, we report the detection of PPV8 in Hungarian and Slovakian pig farms and the estimated prevalence of the virus in Hungary. Altogether, 2230 serum, 233 oral fluid, and 115 processing fluid samples were systematically collected from 23 Hungarian and 2 Slovakian pig farms between 2020 and 2023. A real-time quantitative PCR method was developed to detect the viral genome. Our results revealed the presence of PPV8 on 65% of the Hungarian farms and both Slovakian farms included in our study, marking its first detection in Europe. Oral fluid samples showed the highest positivity rates, reaching up to 100% in some herds. The viral genome was successfully detected in serum and processing fluid samples too, but with significantly lower prevalence rates of 4% and 5%, respectively. Genetic analysis of 11 partial VP2 sequences demonstrated high similarity to the original Chinese strain but with unique amino acid mutations, suggesting possible local evolution of the virus. Our study presents the first scientific evidence of PPV8 infection outside of China and offers a comprehensive assessment of its prevalence in the Hungarian pig population. Further research is required to understand its potential impact on swine health.

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.

Duration of maternally derived antibodies of porcine parvovirus in growing pigs and presence of antibodies in gilts and sows vaccinated with three different parvovirus vaccines.

While gilts and sows are regularly vaccinated against the porcine parvovirus (PPV), little is known on the presence of antibodies in vaccinated sows nor the decline of maternally derived antibodies (MDA) in their offspring. On twelve farms serum samples were taken from 180 gilts and sows vaccinated at least twice with one of three different commercial PPV vaccines. On nine farms, additional 270 serum samples were collected from growing pigs of three different age categories. All 450 samples were examined for PPV antibodies (Abs) by ELISA and haemagglutination inhibition (HI) assay. In total, 65% of all gilts vaccinated twice with either vaccine 1 or vaccine 3 were seronegative by HI assay. In each farm, there were at least three animals with high Ab titres (≥ 1:1280) indicating the presence of PPV in all twelve study farms. However, PPV DNA could not be detected in collected faecal samples. While low to moderately high Ab titres (1:10-1:640) were measured in 98% of twelve-weeks-old pigs, ELISA was only positive in 30% of the same pigs. Though, the statement on the duration of MDA may depend on the applied test, we could confirm an exponential decay of MDA. In addition, we could demonstrate that applied serological tools are insufficient for the confirmation of successful vaccination.

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.

Detection of selected pathogens in reproductive tissues of wild boars in the Campania region, southern Italy.

Monitoring disease among wildlife is critical to preserving health in both domestic animals and wildlife, and it becomes much more critical when the diseases cause significant economic damage to the livestock industry or threaten public health. Given the continuous increase in populations and its role as a reservoir for several infections, wild boar (Sus scrofa) requires special attention regarding disease surveillance and monitoring. In this study, we investigated the molecular prevalence of selected pathogens in the wild boar population of Campania, southern Italy. The prevalence of pathogens causing reproductive problems in pigs (Sus domesticus), including porcine parvovirus (PPV), porcine circovirus types 2 and 3 (PCV-2 and PCV-3), pseudorabies virus (PRV), Coxiella burnetii, and Brucella suis, was evaluated by testing the reproductive organs collected from 63 wild boars with polymerase chain reaction. The most common pathogens were PPV (44.4%) and two porcine circoviruses (14.3%). PRV and C. burnetii, on the other hand, showed a significantly lower prevalence (1.6%). No reproductive organs tested were positive for B. suis. Risk factor analysis revealed a correlation between age and PCV-2 positivity, with animals less than 12 months old having significantly higher prevalence rates.Our findings suggest that wild boars hunted in the Campania region harbour several infections potentially transmissible to other mammals' reproductive tracts. Furthermore, our results emphasized the importance of strict adherence to biosecurity protocols on domestic swine farms, especially on free-range farms, to avoid interactions between domestic and wild animals.

Antiviral alternatives against important members of the subfamily Parvovirinae: a review.

Parvoviruses are responsible for multiple diseases, and there is a critical need for effective antiviral therapies. Specific antiviral treatments for parvovirus infections are currently lacking, and the available options are mostly supportive and symptomatic. In recent years, significant research efforts have been directed toward understanding the molecular mechanisms of parvovirus replication and identifying potential targets for antiviral interventions. This review highlights the structure, pathogenesis, and treatment options for major viruses of the subfamily Parvovirinae, such as parvovirus B19 (B19V), canine parvovirus type 2 (CPV-2), and porcine parvovirus (PPV) and also describes different approaches in the development of antiviral alternatives against parvovirus, including drug repurposing, serendipity, and computational tools (molecular docking and artificial intelligence) in drug discovery. These advances greatly increase the likelihood of discoveries that will lead to potent antiviral strategies against different parvovirus infections.

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.

Expression and self-assembly of virus-like particles from porcine parvovirus and its application in antibody detection.

Porcine parvovirus (PPV) is a major causative agent in reproductive pig disease. The swine industry faces a significant economic and epizootic threat; thus, finding a reliable, quick, and practical way to detect it is essential. In this investigation, recombinant PPV VP2 protein was expressed in the Escherichia coli ( E. coli) expression systems. As shown by electron microscopy (TEM), Western blot, and hemagglutination (HA) assays, the recombinant VP2 protein was successfully assembled into virus-like particles (VLPs) after being expressed and purified. These VLPs had a structure that was similar to that of real PPV viruses and also exhibited HA activity. These VLPs induced high levels of PPV-specific antibody titers in mice after immunization, indicating that the VLPs may be beneficial as potential candidate antigens. VLPs were used as the coating antigens for the VLP ELISA, and the PPV VLPs-based ELISA displayed a high sensitivity (99%), specificity (93.0%) and agreement rate (98.3%) compared to HI assay, and the agreement rate of this ELISA was 97.5% compared to a commercial ELISA kit. Within a plate, the coefficient of variation (CV) was 10%, and between ELISA plates, the CV was 15%. According to a cross-reactivity assay, the technique was PPV-specific in contrast to other viral illness sera. The PPV VLP indirect-ELISA test for PPV detection in pigs with an inactivated vaccine showed that the PPV-positive rate varied among different sample sources from 88.2 to 89.6%. Our results indicate that this ELISA technique was quick, accurate, and repeatable and may be used for extensive serological research on PPV antibodies in pigs.

A Subunit Vaccine Based on the VP2 Protein of Porcine Parvovirus 1 Induces a Strong Protective Effect in Pregnant Gilts.

Porcine parvovirus 1 (PPV1) is one of the most prevalent pathogens that can cause reproductive disorder in sows. The VP2 protein of PPV1 is the most important immunogenic protein that induces neutralizing antibodies and protective immunity. Thus, VP2 is considered an ideal target antigen for the development of a genetically engineered PPV1 vaccine. In this study, the baculovirus transfer vector carrying the HR5-P10-VP2 expression cassette was successfully constructed with the aim of increasing the expression levels of the VP2 protein. The VP2 protein was confirmed using SDS‒PAGE and Western blot analyses. Electronic microscope analysis showed that the recombinant VP2 proteins were capable of self-assembling into VLPs with a diameter of approximately 25 nm. The immunogenicity of the VP2 subunit vaccine was evaluated in pigs. The results showed that VP2 protein emulsified with ISA 201VG adjuvant induced higher levels of HI antibodies and neutralizing antibodies than VP2 protein emulsified with IMS 1313VG adjuvant. Furthermore, the gilts immunized with the ISA 201VG 20 µg subunit vaccine acquired complete protection against PPV1 HN2019 infection. In contrast, the commercial inactivated vaccine provided incomplete protection in gilts. Therefore, the VP2 subunit vaccine is a promising genetically engineered vaccine for the prevention and control of PPV1.

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.

Examination on the Occurrence of Coinfections in Diagnostic Transmittals in Cases of Stillbirth, Mummification, Embryonic Death, and Infertility (SMEDI) Syndrome in Germany.

The stillbirth, mummification, embryonic death, and infertility (SMEDI) syndrome is most commonly associated with porcine parvovirus 1 (PPV1) infections. Little is known about the occurrence of coinfections with SMEDI-associated pathogens and the associations among these pathogens. In our study, we included 40 SMEDI-affected litters from 18 different farms. In total, 158 out of 358 available fetuses from diagnostic transmittals were selected by systematic random sampling and examined for PCV2, PCV3, PPV1, and Leptospira spp. by q-PCR. Results from diagnostic materials showed the following results: in eleven farms, PCV2 was present; in nine farms, PPV1 was present; in five farms, PCV3 was present; and in two farms, Leptospira spp. was present. The detection of Leptospira spp. was significantly associated with a PCV2 coinfection (OR: 26.3; p < 0.001). PCV3 positivity resulted in a reduced probability of detecting PCV2 in the corresponding fetus (OR: 0.078; p = 0.008). Fetal maceration was associated with Leptospira spp. detection (OR: 8.6; p = 0.003), whereas mummification (p = 0.047), reduced crown-rump length (p < 0.001), and bodyweight (p = 0.001) of fetuses were significantly associated with PPV1 and PCV2 coinfection and thus, presumably, a shorter time to death after infection, indicating an enhanced negative effect on the development of fetuses with PCV2 + PPV1 coinfection.

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.

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.