Allele-Specific Dual PCRs to Identify Members of the 27a Cluster of PPV
Abstract: 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 … Show more
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Diagnosztikumfejlesztés a sertés parvovírus 27a törzse ellen
Összefoglalás. A sertés parvovírus (PPV1) súlyos szaporodási zavarokat okoz sertésekben. Az elmúlt két évtizedben egy sajátos, új genotípus jelent meg Európában (27a). Felvetődött, hogy a PPV1-27a klaszter tagjai hátrányosan befolyásolhatják a PPV1 elleni hatékony vakcinázást. 93 GenBankban található részleges vagy teljes PPV1 nukleotid- és fehérjeszekvencia alapján megerősítettük, hogy a 27a klaszter valóban megkülönböztethető a faj más tagjaitól, és 5 jellemző pontmutációt határoztunk meg. A genetikai különbségek alapján kifejlesztettünk egy kettős allélspecifikus polimeráz láncreakciót a 27a klaszter tagjainak más PPV1 törzsektől való egyszerű és gyors megkülönböztetésére. Az érzékenyítés és a felhasználóbarátabbá tétel érdekében a módszert pedig továbbfejlesztettük qPCR alkalmazásra. Summary. Porcine Parvovirus (PPV) is a significant infectious agent responsible for severe reproductive failure in pigs. Until the 2000s, there was limited systematic study of genetic changes in the PPV genome, as it was believed to be highly immunologically stable. Vaccines developed from “ancient” strains were thought to provide comprehensive protection against all PPV variants. However, in the past two decades, a novel genotype, PPV-27a, has emerged in Europe, becoming the prototype of a distinct genetic cluster. Concerns were raised that members of the PPV-27a cluster might negatively impact effective vaccination against PPV. Accurate identification and quantification of 27a viruses are crucial for understanding the biological significance of these variants. To provide an updated and reliable definition of 27a, 93 databank-deposited nucleotide and protein sequences of the VP2 of various PPV isolates were aligned. It was confirmed that the 27a cluster could be distinguished from other species members, though some divergences were noted compared to earlier defined genetic markers. Phylogenetic analysis revealed that five closely linked point mutations (261C, 682G, 1240T, 1255C, and 1306A) differentiate cluster members from other PPV variants. Based on these genetic differences, a dual allele-specific polymerase chain reaction (PCR) was developed to easily and quickly differentiate 27a cluster members from other PPV strains. Two of the defined point mutations (261C and 682G) were utilized to create an allele-specific primer set (PS1) for the development of a 27a-specific asPCR system. The dual PCR had a detection limit of <1.66 × 104 copies/reaction. To enhance sensitivity and user-friendliness, the method was adapted for quantitative PCR (qPCR) with fluorescent probes. The sensitivity improvement of the method was approximately two logs (<1.66 × 104 copies/reaction for dual PCR versus <2.40 × 102 copies/reaction for dual qPCR). To validate the PCR method, clinical samples were collected from cases of reproductive failure involving various types of fetal losses, including mummified or aborted fetuses. Both the dual PCR and dual qPCR were used to distinguish 27a and non-27a PPV viruses in these field samples. Thirty-one samples were investigated and from these twenty-six were found to be 27a positive with at least one of the methods. Remarkably, in eight of the fourteen cases, 27a-type viruses were detected, indicating a significant presence of these viruses in the samples. The use of allele-specific PCR primers allows for the rapid differentiation of 27a-type viruses from other PPV genotypes. Depending on user preference, any of the PCR methods developed based on these findings can be utilized as diagnostic tools in veterinary practice. Additionally, this versatile PCR system’s application can facilitate field studies contributing to a clear assessment of virus prevalence and a better understanding of 27a biology.
Diagnosztikumfejlesztés a sertés parvovírus 27a törzse ellen
Összefoglalás. A sertés parvovírus (PPV1) súlyos szaporodási zavarokat okoz sertésekben. Az elmúlt két évtizedben egy sajátos, új genotípus jelent meg Európában (27a). Felvetődött, hogy a PPV1-27a klaszter tagjai hátrányosan befolyásolhatják a PPV1 elleni hatékony vakcinázást. 93 GenBankban található részleges vagy teljes PPV1 nukleotid- és fehérjeszekvencia alapján megerősítettük, hogy a 27a klaszter valóban megkülönböztethető a faj más tagjaitól, és 5 jellemző pontmutációt határoztunk meg. A genetikai különbségek alapján kifejlesztettünk egy kettős allélspecifikus polimeráz láncreakciót a 27a klaszter tagjainak más PPV1 törzsektől való egyszerű és gyors megkülönböztetésére. Az érzékenyítés és a felhasználóbarátabbá tétel érdekében a módszert pedig továbbfejlesztettük qPCR alkalmazásra. Summary. Porcine Parvovirus (PPV) is a significant infectious agent responsible for severe reproductive failure in pigs. Until the 2000s, there was limited systematic study of genetic changes in the PPV genome, as it was believed to be highly immunologically stable. Vaccines developed from “ancient” strains were thought to provide comprehensive protection against all PPV variants. However, in the past two decades, a novel genotype, PPV-27a, has emerged in Europe, becoming the prototype of a distinct genetic cluster. Concerns were raised that members of the PPV-27a cluster might negatively impact effective vaccination against PPV. Accurate identification and quantification of 27a viruses are crucial for understanding the biological significance of these variants. To provide an updated and reliable definition of 27a, 93 databank-deposited nucleotide and protein sequences of the VP2 of various PPV isolates were aligned. It was confirmed that the 27a cluster could be distinguished from other species members, though some divergences were noted compared to earlier defined genetic markers. Phylogenetic analysis revealed that five closely linked point mutations (261C, 682G, 1240T, 1255C, and 1306A) differentiate cluster members from other PPV variants. Based on these genetic differences, a dual allele-specific polymerase chain reaction (PCR) was developed to easily and quickly differentiate 27a cluster members from other PPV strains. Two of the defined point mutations (261C and 682G) were utilized to create an allele-specific primer set (PS1) for the development of a 27a-specific asPCR system. The dual PCR had a detection limit of <1.66 × 104 copies/reaction. To enhance sensitivity and user-friendliness, the method was adapted for quantitative PCR (qPCR) with fluorescent probes. The sensitivity improvement of the method was approximately two logs (<1.66 × 104 copies/reaction for dual PCR versus <2.40 × 102 copies/reaction for dual qPCR). To validate the PCR method, clinical samples were collected from cases of reproductive failure involving various types of fetal losses, including mummified or aborted fetuses. Both the dual PCR and dual qPCR were used to distinguish 27a and non-27a PPV viruses in these field samples. Thirty-one samples were investigated and from these twenty-six were found to be 27a positive with at least one of the methods. Remarkably, in eight of the fourteen cases, 27a-type viruses were detected, indicating a significant presence of these viruses in the samples. The use of allele-specific PCR primers allows for the rapid differentiation of 27a-type viruses from other PPV genotypes. Depending on user preference, any of the PCR methods developed based on these findings can be utilized as diagnostic tools in veterinary practice. Additionally, this versatile PCR system’s application can facilitate field studies contributing to a clear assessment of virus prevalence and a better understanding of 27a biology.
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