Porcine Parvovirus Strains Research Articles

Isolation and characterization of porcine parvovirus in Vietnam.

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). 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. 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. 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.

A Plant-Produced Porcine Parvovirus 1-82 VP2 Subunit Vaccine Protects Pregnant Sows against Challenge with a Genetically Heterologous PPV1 Strain

Porcine parvovirus (PPV) causes reproductive failure in sows, and vaccination remains the most effective means of preventing infection. The NADL-2 strain has been used as a vaccine for ~50 years; however, it does not protect animals against genetically heterologous PPV strains. Thus, new effective and safe vaccines are needed. In this study, we aimed to identify novel PPV1 strains, and to develop PPV1 subunit vaccines. We isolated and sequenced PPV1 VP2 genes from 926 pigs and identified ten PPV1 strains (belonging to Groups C, D and E). We selected the Group D PPV1-82 strain as a vaccine candidate because it was close to the highly pathogenic 27a strain. The PPV1-82 VP2 protein was produced in Nicotiana benthamiana. It formed virus-like particles and exhibited a 211 agglutination value. The PPV1-190313 strain (Group E), isolated from an aborted fetus, was used as the challenging strain because it was pathogenic. The unvaccinated sow miscarried at 8 days postchallenge, and mummified fetuses were all PPV1-positive. By contrast, pregnant sows vaccinated with PPV1-82 VP2 had 9-11 Log2 antibody titers and produced normal fetuses after PPV1-190313 challenge. These results suggest the PPV1-82 VP2 subunit vaccine protects pregnant sows against a genetically heterologous PPV1 strain by inducing neutralizing antibodies.

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.

Viral Fitness and Antigenic Determinants of Porcine Parvovirus at the Amino Acid Level of the Capsid Protein.

Since 2001, strains of porcine parvovirus (PPV), designated 27a-like strains, were observed in Europe, suggesting a predominance of these viruses over older strains. The reasons for the obvious evolutionary advantage are unknown. Here, a series of mutants containing amino acid replacements found in the predominant field strains were generated in a PPV-NADL2 background, and their impact on replication efficiency and antibody binding activity was determined. Some amino acid substitutions observed in the 27a-like strains significantly increased viral fitness and decreased neutralization activity of serum samples raised against commercial vaccines and old virus strains (e.g., NADL2). These mutant viruses and a monoclonal antibody raised against a classical PPV strain defined a 27a-specific neutralizing epitope around amino acid 228 of the capsid protein VP2. Based on the analysis of the mutant viruses, it is hypothesized that the predominant factor for the global spread of the PPV-27a strain substitutions is an increased viral fitness of the 27a-like viruses, possibly supported by partial immune selection. This is reminiscent to the evolution of canine parvovirus and worldwide replacement of the original virus by the so-called new antigenic types. IMPORTANCE Porcine parvovirus is one of the most important causes of reproductive failure in swine. Recently, despite the continuous use of vaccines, "new" strains emerged, leading to the hypothesis that the emergence of new amino acid substitutions could be a viral adaptation to the immune response against the commercial vaccines. Our results indicate the amino acid substitutions observed in the 27a-like strains can modify viral fitness and antigenicity. However, an absolute immune escape was not evident.

An octavalent vaccine provides pregnant gilts protection against a highly virulent porcine parvovirus strain

BackgroundPorcilis® Ery+Parvo+Lepto is an octavalent inactivated ready-to-use vaccine that contains Erysipelothrix rhusiopathiae (Ery), porcine parvovirus (PPV), and six serogroups of Leptospira (Lepto). The efficacy of Porcilis® Ery + Parvo+Lepto against reproductive problems associated with porcine parvovirus (PPV) infection was evaluated in pregnant gilts. For this, a group of ninegilts was vaccinated twice (at 5 and 6 months old) with Porcilis® Ery + Parvo+Lepto (Group 1), while a group of eight gilts was included as unvaccinated controls (Group 2). All pigs were artificially inseminated 4 weeks after the second vaccination. They were challenged during early gestation with PPV-27a, a virulent cluster D strain, and euthanized to collect their offspring by hysterectomy around day 90 in pregnancy. Antibody responses against PPV in gilts were measured, and the presence of PPV in progeny was also determined.ResultsNo clinical signs were observed after vaccination. After PPV challenge, all foetuses from the vaccinated gilts were alive (132/132), while in the unvaccinated group only 41% were alive (46/112), 19.6% were dead and 39.4% of the offspring (44/112) were mummified. PPV could be detected by qPCR in 14% of the progeny from vaccinated gilts at an average of 4.7 log10/ml, whereas this was significantly higher in the control group, where 90% of the progeny were PPV positive, with titres of 9.8 log10/ml on average.ConclusionsThe present study demonstrates that vaccination of gilts with Porcilis® Ery + Parvo+Lepto was safe and induced an immune response sufficient to protect progeny against PPV by reducing transplacental infection.

Identification and characterization of porcine parvovirus in Hunan province, China

Porcine parvovirus (PPV) is considered one of the most important infectious agents of reproductive failure in sows. Little information, however, is available on its prevalence in healthy fattening pigs. Therefore, in the present study, 197 fecal swabs, 197 nasal swabs, 389 serum samples, and 310 lung samples were collected from pigs aged 10-25 weeks across Hunan, China, and tested for the presence of PPV. PPV DNA was amplified by polymerase chain reaction, demonstrating an overall positivity rate of 7.69%, with a particularly high infection rate of 22.90% in the lungs. A total of five PPV strains (PPV-HuN1-5) were isolated on the basis of cytopathic effects in swine testicular cells, and the near-complete genomes were sequenced and subjected to phylogenetic analysis with reference to reported PPV sequences in GenBank. The five Hunan isolates showed a close relationship with each other and predominantly with reported European PPV strains. Moreover, seven amino acid substitutions were detected within the coding region of the VP2 of PPV-HuNs when compared with that of the Chinese vaccine strain PPV-NJ. The relatively high prevalence of PPV discovered in healthy fattening pigs despite a long-term vaccination program in China, highlights the need for improved prevention, monitoring, and control of related diseases in herds.

Detection and molecular characterization of porcine parvovirus in fetal tissues from sows without reproductive failure in Argentina

Porcine parvovirus (PPV) is one of many pathogens responsible for reproductive failure in pregnant sows. Several studies have reported the appearance of new PPV strains that differ from previous isolates both genetically and antigenically. Thus, the protective effects of commercially inactivated vaccines could not be complete. In South America, the information about PPV is limited. Thus, the aim of the present study was to detect and characterize the PPV strains present in 131 mummies or stillbirths from normal deliveries in sows from a commercial swine farm of Argentina that uses the commercial vaccine. PCR results showed that 17/131 were positive to PPV. Ten of these viruses were isolated and sequenced. All viruses were related to the PPV1 sequence (NADL-2), maintaining the amino acid differences in positions 436 (S–P) and 565 (R–K). This study is the first to report the isolation of PPV in Argentina and the results suggest that PPV can cross the placenta even in vaccinated sows, thus affecting some of the fetuses and being able to cause fetal death in sows without reproductive failure. The results also suggest that vaccination only reduces clinical signs and reproductive disorders and may thus not be a perfect tool to manage PPV infection. This study provides information that needs to be studied in depth to improve strategies to prevent and control PPV infection in swine farms.

Estimating the prevalence of antibodies against Ungulate protoparvovirus 1 (porcine parvovirus) in wild boar populations for Saxony, Germany

Despite the high population density of wild boars ( Sus scrofa) in Germany and the pathogene importance of Porcine parvovirus (PPV), only few data are available about the presence of PPV within this population. Here, the presence of PPV antibodies in sera of the wild boar population were determined in Saxony, Germany, an area of swine production and a natural habitat for wild boar. As a result, 155/285 samples were positive (titer ≥80), and the majority (89.68%) of these positive samples had titers ranging between 320 and 40,960. Increased titers were generally observed in older animals (without statistical significance), but no differences in sex and hunting sites could be determined. These results suggest that the PPV infection rate in Germany (at least in Saxony) is higher than those reported for most other European regions. Since wild boars can harbor new PPV types, these animals need to be intensively monitored in order to avoid the introduction of new PPV strains in the domestic swine population.

Isolation and phylogenetic analysis of a new Porcine parvovirus strain GD2013 in China

Porcine parvovirus (PPV), a causative agent of an infectious reproductive disorder causing stillbirth, mummification, embryonic death and infertility (SMEDI) syndrome in swine, is a threat to both domestic pigs and wild boars regardless of age and gender. Recent studies found that the observed average substitution rate in the PPV genome was close to those of the RNA viruses and new strains showing serological neutralization activities different from that of the vaccine strain NADL-2 have been reported. These observations have increased the need for the development of new commercial vaccine strains.In this study, a new PPV strain, GD2013, was isolated from Guangdong, China, and its entire genome sequenced. A phylogenetic tree based on the complete coding region of the genomes of 32 PPV strains was constructed using the Bayesian Markov Chain Monte Carlo (MCMC) method. The results showed that strain GD2013 fell into the same phylogenetic cluster as the classical vaccine strains NADL-2 and POVCAP, suggesting a close relationship to the vaccine strains. Multiple sequence alignments and amino acid mutation analyses of the PPV VP2 gene revealed a new amino acid polymorphism site at Thr45 on VP2 that could be used to identify low virulence strains as vaccine candidates. Selective pressure analysis of the NS1 and VP2 genes by calculating the mean rates of non-synonymous substitutions (dN) over synonymous substitutions (dS) implied that both of these genes were under negative selection. Therefore, by using phylogenetic and amino acid mutation analyses, a likely candidate strain suitable for evaluation as an attenuated vaccine strain was identified.

Construction and characterization of the infectious clone of porcine parvovirus carrying genetic marker

Porcine parvovirus (PPV) is one of the major pathogens that bring about reproductive failure of pregnant sows. However, the study of the pathogenesis mechanism is circumscribed due to the lack of efficient genetic manipulation method. Infectious clone is a powerful tool for further studying the genetic mechanisms of PPV. In the present study, the gene fragment (157–4812) of PPV was amplified by PPV China isolate strain as a template, and PPV DNA fragments (1–182) forming Y-structure within in 5′ end and (4788–5074) forming U-structure in 3′ end were synthesized. And then, the above three fragments were inserted into plasmid pKQLL to congregate a PPV full-length recombinant plasmid by means of In-Fusion cloning technology. After the successful sequencing identification of the recombinant plasmid, the EcoR I restriction site was brought out as a genetic marker by nonsense mutation (A3058 T) to produce plasmid Y-PPV, which was transfected into PK-15 cells for rescue of virus. The rescued viral particles were observed under transmission electron microscopy, and the sequencing analysis showed that Y-PPV could stably carry the genetic marker. It could be seen that Y-PPV has similar replicate capability and pathogenicity as the wild-type parental PPV strain by cellular and animal experiments. These results confirmed that Y-PPV maintain similar biological characteristics with wild-type parental PPV strain. Infectious clone could be a valuable tool for studying the individual genes of PPV and applications in gene deletion or live vector vaccines.

TESTING OF COMBINED VACCINES AGAINST AUJEZSKY’S DISEASE, PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME AND PORCINE PARVOVIRUS INFECTION FOR THEIR ANTIGENICITY

Tests of inactivated emulsion anti-Aujezsky’s disease, porcine reproductive and respiratory syndrome (PRRS) vaccine based on marker Aujezsky’s disease virus strain, PRRS virus strain and inactivated emulsion anti-Aujezsky’s disease and porcine parvovirus infection vaccine based on marker Aujezsky’s disease virus strain and porcine parvovirus strain developed by the FGBI “ARRIAH” for their antigenicity are described in the paper. It is demonstrated that the said vaccines can be used successfully for PRRS and porcine parvovirus infection and Aujezsky’s disease prevention. They are becoming of special significance when used in programs on Aujeszky’s disease control and eradication. The use of the vaccine based on marker Aujeszky’s disease virus strain allows differentiation between the animals that have antibodies to the marker vaccine strain (in the absence of antibodies to the glycoprotein gE in 100% of cases) from the animals with antibodies to field strains of the Aujeszky’s disease virus in their sera. A comprehensive assessment of the use of associated vaccines developed in the FGBI “ARRIAH” (against Aujeszky’s disease based on the marker strain, reproductive and respiratory syndrome, parvovirus infection), in the framework of anti-epidemic measures on a pig farm of the Russian Federation showed their safety, high antigenicity, immunogenicity.

Perspectives on the Evolution of Porcine Parvovirus.

Porcine parvovirus (PPV) is one of the main causes of porcine reproductive failure. It is important for swine industries to understand the recent trends in PPV evolution. Previous data show that PPV has two genetic lineages originating in Germany. In this study, two more genetic lineages were defined, one of which was distinctly Asian. Additionally, amino acid substitutions in European strains and Asian strains showed distinct differences in several regions of the VP2 gene. The VP1 gene of the recent PPV isolate (T142_South Korea) was identical to that of Kresse strain isolated in the USA in 1985, indicating that modern PPV strains now resemble the original strains (Kresse and NADL-2). In this study, we compared strains isolated in the 20th century to recent isolates and confirmed the trend that modern strains are becoming more similar to previous strains.

An inactivated whole-virus porcine parvovirus vaccine protects pigs against disease but does not prevent virus shedding even after homologous virus challenge.

Inactivated whole-virus vaccines against porcine parvovirus (PPV) can prevent disease but not infection and virus shedding after heterologous virus challenge. Here, we showed that the same is true for a homologous challenge. Pregnant sows were vaccinated with an experimental inactivated vaccine based on PPV strain 27a. They were challenged on day 40 of gestation with the virulent porcine parvovirus PPV-27a from which the vaccine was prepared (homologous challenge). On day 90 of gestation, the fetuses from vaccinated sows were protected against disease, while the fetuses of the non-vaccinated sows (control group) exhibited signs of parvovirus disease. All gilts, whether vaccinated or not vaccinated, showed a boost of PPV-specific antibodies indicative of virus infection and replication. Low DNA copy numbers, but not infectious virus, could be demonstrated in nasal or rectal swabs of immunized sows, but high copy numbers of challenge virus DNA as well as infectious virus could both be demonstrated in non-vaccinated sows.

Establishment and preliminary application of an assay for the detection of porcine parvovirus in cells used for production

Objective To establish an assay for the detection of porcine parvovirus (PPV) and to verify its application for monitoring cells used for production. Methods A pair of primers and one probe were designed according to the conserved sequence encoding non-structural protein 1 (NS1). Based on the designed primers, a real-time fluorescent quantitative PCR assay for the detection of PPV was developed. Several parameters including the linearity, precision, minimum detection limit and anti-interference of the established assay were evaluated. A stock of PPV strains was prepared by infecting swine testicle (ST) cells with PPV strains. An assay for the detection of PPV infection was developed by using ST cells as sensitive cells. A combined ST cell infection-PCR test was developed by combining the ST cell infection assay with the real-time fluorescent quantitative PCR assay. The sensitivity of ST cell infection-PCR test was analyzed. The cell samples used for production of biological products were detected by using the established assay. Results The real-time fluorescent quantitative PCR assay was specific for the detection of PPV without cross-reaction to other species of parvovirus virus, SV40 virus and other porcine viruses. The linear range of the assay was 1×109-1×104 copies/μl with a R2 value more than 0.98. The sensitivity of the real-time quantitative PCR assay was 1×104 copies/μl. Both of the intra- and inter-coefficient of variation (CV) were less than 5% in Ct values. The intra- and inter-CV in copies of detection were 5%-15% and 30%-40% respectively. The minimum detection limit of the real-time fluorescent quantitative PCR assay was 1CCID50/ml. The PPV strains were detected in cell samples with no interference. The sensitivity of ST cells infection-PCR test was 0.01CCID50/ml. All of the 22 cell samples were negative for PPV by using the real-time fluorescent quantitative PCR assay. Conclusion The real-time fluorescent quantitative PCR and the ST cell infection-PCR test for the detection of PPV in cells were established successfully. The application of the two assays was conducive to further enhance the safety of using cells for production and therapy. Key words: Porcine parvovirus (PPV); Real-time fluorescent quantitative PCR assay; Swine testicle (ST) cells; Infection assay; Infection-PCR assay

Differential replication of two porcine parvovirus strains in bovine cell lines ensues from initial DNA processing and NS1 expression

Porcine parvovirus (PPV) is a small DNA virus with restricted coding capacity. The 5 kb genome expresses three major non-structural proteins (NS1, NS2 and SAT), and two structural proteins (VP1 and VP2). These few viral proteins are pleiotropic and interact with cellular components throughout viral replication. In this regard, very few cell lines have been shown to replicate the virus efficiently. Cell lines were established from a primary culture of bovine cells that allowed allotropic variants of PPV to be distinguished. Three cell lines were differentially sensitive to infection by two prototype PPV strains, NADL-2 and Kresse. In the first cell line (D10), infection was restricted early in the infectious cycle and was not productive. Infection of the second cell line (G11) was 1000 times less efficient with the NADL-2 strain compared with porcine cells, while production of infectious virus of the Kresse strain was barely detectable. Restriction points in these cells were the initial generation of DNA replication intermediates and NS1 production. Infection with chimeras between NADL-2 and Kresse showed that residues outside the previously described allotropic determinant were also partially responsible for the restriction to Kresse replication in G11 cells. F4 cells were permissive to both strains, although genome replication and infectious virus production were lower than in the porcine cells used for comparison. These results highlight the dependent nature of parvovirus tropism on host factors and suggest that cells from a non-host origin can fully support a productive infection by both strains.

Complete Genome Sequence of a Porcine Parvovirus Strain Isolated in Central China

We report here the complete genome sequence of the porcine parvovirus (PPV) strain J-PPV, isolated from central China. Our data, together with sequence data for PPV isolates from other regions of China, will help in understanding the epidemiology and molecular characteristics of PPV field isolates in China.

Phylogeny and evolution of porcine parvovirus

Porcine parvovirus (PPV), a member of the genus Parvovirus, family Parvoviridae, is a significant causative agent in porcine reproductive failure, causing serious economic losses in the swine industry. Previous phylogenetic studies based on the NS1 or VP2 genes indicated that current PPV strains diverged 30 years ago and that VP2 was under neutral or positive selection. Our analysis of NS1, VP2 and complete ORFs indicated that the most recent common ancestor of PPV strains existed about 250 years ago and that the 127-nt repeat in the 3′NTR was present in viruses of some subclades that evolved about 80 years ago. Nucleotide substitution rates of NS1 and VP2 genes were 3.03×10−5 and 1.07×10−4, respectively. Both the NS1 and VP2 proteins were under purifying selection and recombination did not contribute to the genetic diversity of PPV. As expected, surface amino acids are hydrophilic and make up the majority of mutations in the VP2 protein; residues in VP2 interfaces were substituted gradually, often in conjunction with complementary substitutions in the neighboring VP2.

Characterization of the capsid protein VP2 gene of a virulent strain NE/09 of porcine parvovirus isolated in China

A virulent strain NE/09 of porcine parvovirus (PPV) was isolated from mummified swine fetus samples in China and its capsid protein VP2 gene was analyzed. The data showed that the VP2 gene encompassed an open reading frame of 1713 nucleotides with a composition of 684 adenine (39.9%), 387 cytosine (22.6%), 268 guanine (15.7%), and 374 thymine (21.8%). The VP2 gene encoded a 570aa-long protein which could be recognized by anti-PPV-VP2 monoclonal antibody 3C9. Sequence analysis revealed that the VP2 gene of PPV-NE/09 had close sequence similarity with the VP2 gene of other PPV strains. However, a 27 nucleotide region that encodes a glycine-rich domain at the N-terminal region of VP2 was deleted in the PPV-NE/09 strain. The PPV-NE/09 virions were purified by 1.39g/ml CsCl density centrifugation and subjected to SDS–PAGE analysis. The results showed that only two major polypeptides VP1 and VP2 with molecular weights of 83 and 64kDa, respectively were observed, whereas VP3 with a molecular weight of 60kDa was not present, in contrast to the PPV-NADL-2 strain. According to the phylogenetic relationship of the VP2 gene of PPV-NE/09, the isolate was a new mutant strain of PPV prevailing in China.

Phylogeny and evolutionary genetics of porcine parvovirus in wild boars

Porcine parvovirus (PPV) is widespread among swine and is responsible for reproductive failure of susceptible sows, characterized by embryonic and fetal death. Studies showed that PPV in domestic pig is genetically diverse and some strains differ from the ones used for vaccination. Organ samples from wild boars and domestic pigs were collected in Transylvania (Romania) and tested for the presence of PPV by polymerase chain reaction. Positive samples were grouped and 14 from the wild boar and 1 from the domestic pig PPVs were selected for VP1/VP2 sequence analysis and comparison with available GenBank data. The molecular clock analysis revealed that PPV has a relatively recent evolutionary history, originated approximately 120years ago and the main divergence occurred in the last 20–60years. Phylogenetic and residue substitution analysis showed that the viruses could be divided into 6 distinct clusters and that wild boar PPVs were partially different and independent from domestic pig PPVs. PPVs of wild boars proved to be more diverse than viruses of domestic pigs. The presence of the highly virulent 27a-like PPV strains in wild boars was also detected.

Establishment of a porcine parvovirus (PPV) DNA standard and evaluation of a new LightCycler nested-PCR assay for detection of PPV

Porcine parvovirus (PPV) is a major causative agent in a syndrome of reproductive failure in swine. In validation (viral clearance) studies, PPV is a model of non-enveloped viruses and is widely used instead of human parvovirus B19, which causes a variety of illnesses including erythema infectiosum (fifth disease) in children and hydrops fetalis in pregnant women. To improve the sensitivity of current PCR-based assays for detection of PPV and to standardize the quantification of PPV, we have developed a lightcycler (LC) nested-PCR (nPCR) assay and constructed a PPV DNA standard evaluated in the LC nPCR assay. The PPV DNA standard, a plasmid termed pPPV, encodes a 3.3 kb PPV NADL-2 genome fragment. One genome copy equivalent (gce) of PPV equals 6.7 attograms of pPPV. The LC nPCR assay is a simple and specific method developed for detection of PPV strains but not any other viruses including members of Parvoviridae. The first 25-cycle PCR with outer primers chose by comparative analysis of 12 primers in 21 different combinations and a second 45-cycle PCR with inner primers amplify 286 and 251 bp fragments of PPV genome, respectively, for 40 min with a sensitivity of approximately 100 gce per assay (ml). By using the LC nPCR assay for analysis of PPV samples with known infectivity, we found that one 50% tissue culture infectious dose (TCID 50) equals 1.93±0.24 log 10 gce.