Pathomolecular Studies of Porcine Parvovirus in Assam

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

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.

Porcine parvovirus (PPV) infection induces germ cell death, leading to reproductive disorders in first-pregnant sows. Porcine placental trophoblast cells (PTCs) are the major target of PPV, and we have previously found that PPV infection leads to the death of PTCs by a non-apoptotic process, which may be related to PPV pathogenicity. The Z-nucleic acid-binding protein 1 (ZBP1) is often activated after virus invasion and mediates subsequent cell death. Here, we found that PPV infection induced ZBP1-mediated necroptosis of porcine PTCs in the presence of the apoptosis inhibitor, AC-DEVD-CHO. ZBP1 expression was upregulated during PPV infection, and ZBP1 knockout or RNA interference significantly reduced its expression along with the PPV-induced necroptosis. We discovered that RIPK3 and MLKL, but not Caspase-8, were involved in downstream signaling of ZBP1 during PPV infection; the phosphorylation levels of RIPK3 and MLKL were enhanced, but Caspase-8 was not significantly cleaved. The knockout of RIPK3 and MLKL significantly reduced the PPV infection-induced necroptosis of porcine PTCs. RIPK3 knockout did not affect the PPV infection-induced upregulation of ZBP1 expression, but blocked the activation of MLKL. MLKL knockout did not affect the upregulation of ZBP1 expression and RIPK3 phosphorylation during PPV infection. UV-inactivated PPV induced significantly less necroptosis of porcine PTCs than non-irradiated PPV. A PPV strain with a mutation in the translation initiation codon was still able to induce necroptosis of PTCs through the ZBP1/RIPK3/MLKL pathway. These results provide new insights into the pathogenic mechanism of PPV infection and suggest that PPV infection of porcine PTCs induces necroptosis through the viral DNA-dependent ZBP1/RIPK3/MLKL pathway.

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

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

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

Phylogeny and evolution of porcine parvovirus

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.

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.

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.

Simple SummaryWild animals can transmit diseases to domestic animals. In Africa, warthogs are known to be carriers of pathogens that can infect pigs; consequently, it is important to identify these pathogens in order to protect pigs from infection. In this study, two important swine pathogens i.e., African swine fever virus (ASFV) and porcine parvovirus 1 (PPV1) were identified in warthogs in Namibia and characterized genetically. The results will be of interest to those working in swine disease management and control in Namibia.Understanding virus circulation in wild animals, particularly those that have contact with domestic animals, is crucial for disease management and control. In Africa, warthogs are known to be asymptomatic carriers of porcine pathogens; a recent study in Namibia has shown them to be positive for Porcine circovirus-2 (PCV-2). In this study, the same samples used for the PCV-2 investigation in Namibia were further screened for the presence of African swine fever virus (ASFV) and porcine parvovirus 1 (PPV1) by PCR. Of the 42 animals tested, 2 (4.8%) and 13 (31%) were positive for AFSV and PPV1, respectively. The two AFSV were also co-infected with PPV1. Combing the results of this study with the results of the previous PCV-2 investigation, four warthogs were shown to be co-infected with both PPV1 and PCV-2. Sequence and phylogenetic analysis revealed that the AFSV belonged to genotype (Ib) but were from different serogroups. Unexpectedly, the ASFVs from the warthogs were genetically distinct to those observed in an outbreak in the same region of Namibia that occurred less than fifteen months prior to the sampling of the warthogs. In fact, a stronger genetic relationship was observed between the warthog viruses and historical Namibian and South African ASFVs identified in 1980, 2004 and 2008. For the PPV1s, the closest relative to the Namibian PPV1 were viruses identified in wild boar in Romania in 2011. This study confirms that warthogs are carriers of porcine pathogens and the data should encourage further studies on larger populations of wild and domestic swine to more fully understand the epidemiology and transmission of viral pathogens from these species.

The present study was undertaken to detect the presence of porcine circovirus type 2 (PCV2) and porcine parvo virus (PPV) in 54 porcine aborted foetuses, stillborns and mummified foetuses. Foetal tissues of heart, lung, spleen and lymphnodes were used for extraction of total DNA and the presence of viral pathogens were confirmed through polymerase chain reaction (PCR) technique. Out of 54 samples, nine (16.6%) samples were positive for PCV2, eight (14.8%) samples were positive for PPV while co-infection with PCV2 and PPV was detected in four (7.4%) samples. The histopathological changes in the foetuses were found to be mostly necrosis in the cells of the developing organs viz. lungs, kidney, liver, heart, brain, spleen and lymphnodes. Lymphoid depletion was observed in the lymphoid follicles of the lymphnodes. Meningoencephalitis in the cerebrum was also seen in both PPV and PCV2 infected foetuses. The results indicated that PPV and PCV2 could be an important infectious agent in cases of porcine stillbirths, abortions and mummified foetuses.

Porcine parvovirus (PPV) is a DNA virus and causative agent of several reproductive problems in sows. This study was conducted to determine the prevalence and analyze the DNA sequence of structural protein encoding gene (VP) of PPV2 genotype in pigs. A total of 146 samples (lung and blood samples) were collected from slaughtered pigs of seven provinces in Central Vietnam during 2018-2019. The overall prevalence of PPV2 was 56.2% (82/146). PPV2 positive rate in each province ranged from 37.5% for Quang Nam to 100% for Quang Binh, with the exception of Da Nang, where no PPV2 positive samples were detected. Nearly complete PPV2-VP gene sequences of three strains were identified with the length of 2,493 nucleotides and deposited in GenBank with accession numbers of OL913365-OL913367. Four nucleotide substitutions were detected in Vietnamese PPV2 isolates and were not observed in PPV2 reference strains. Multiple alignment and comparison of nucleotide and deduced amino acid sequences showed the high similarity within Vietnamese PPV2 strains (95.6-96.5% and 94.7-96.9%, respectively). The PPV2 strains from this study clustered together with the "primitive" PPV2 strains from Myanmar, and strains from China in a main clade in the phylogenetic tree (Cluster A). This is the first report on the prevalence of PPV2 genotype and its VP gene sequence in pigs in Vietnam. This also provides the valuable information on the molecular evolution of locally circulating PPV2 and contributes to the control of PPV-induced SMEDI syndrome in sows, especially in the central provinces of Vietnam.

A novel porcine parvovirus DNA-launched infectious clone carrying stable double labels as an effective genetic platform

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.