Diagnosztikumfejlesztés a sertés parvovírus 27a törzse ellen
Authors: Tamás Vivien , Kiss István , Homonnay G. Zalán , Mészáros István , Olasz Ferenc , Zádori Zoltán 
Published: May 2024

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.