Coatomer protein COPƐ, a novel NS1-interacting protein, promotes the replication of Porcine Parvovirus via attenuation of the production of type I interferon.

Porcine Parvovirus (PPV) is a pathogen causing porcine reproductive disorders. Non-structural protein NS1 appears diverse functions acting as a predominant regulator in promoting PPV replication. In this study, we identified a PPV NS1 binding protein coatomer subunit epsilon (COPƐ), and found that COPƐ is a critical regulator during PPV replication. In NS1 transfected or PPV infected cells, COPƐ was interacted with NS1 and translocated into nucleus together with NS1. Knockout of COPƐ could inhibit PPV production by increasing the expression levels of IFN-ß, while overexpression of COPƐ enhanced PPV production by reducing the expression levels of IFN-ß. Furthermore, the domain mapping assay showed that the N-terminal amino acids domain of NS1 (25-EAFSYVF-31) were required for the interaction of COPƐ with NS1. Sequence alignment result displays that parvovirus NS1 (EAFSYVF) amino acids domain is highly conservative among PPV, CPV, FPV and MEV, and down-regulation of COPƐ could also significantly reduce the replication of these viruses. Notably, we found that the interaction of COPƐ with NS1 play an important role in promoting the production of type I interferon during PPV or CPV infection, which affect the replication of these viruses. Taken together, the results presented here show a novel function of NS1 interaction with COPƐ that regulates the parvovirus replication through modulating the type I interferons signaling pathway, provided a potential target for the control of parvovirus-associated diseases.

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

BACKGROUND: Porcilis® 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. RESULTS: No 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. CONCLUSIONS: The 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.

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

Porcine parvovirus (PPV) is one of the major pathogens causing reproductive failure of swine. However, its specific pathogenesis has not been fully elucidated. Infectious clone is a powerful tool for further studying the pathogenic mechanism of PPV. In the present study, a PPV infectious clone was constructed, and the clone carries His-tag and Flag-tag double-genetic marker at the end of the ns1 gene 3' terminal and vp1 gene 5' terminal, respectively. The PPV DNA fragment F1 (1-182) in 5' end and the other PPV DNA fragment F2 (4788-5074) in 3' end were synthesized and assembled to the lower copy plasmid to construct pKQLL(F1 + F2), while the PPV DNA genome as a template to amplify carrying tags sequence PPV middle DNA fragment F3 and F4 by introducing Flag and His tags sequence in primers. Subsequently, the fused fragment F3/F4 were cloned into the Stu I/Sna B I sites of pKQLL(F1 + F2) plasmid to assemble the complete full-length PPV DNA recombinant plasmids, named as pD-PPV. The pD-PPV was transfected into PK-15 cells to gain rescued PPV virus, designed as D-PPV. Moreover, D-PPV showed similar replicate capability and pathogenicity comparing to the wild-type parental PPV through in vitro and in vivo studies, and the double labels can effectively indicate the expression and localization of viral proteins. Finally, the rescued D-PPV was found to be a convenient tool for antiviral drug screening. These data indicated that the newly established reverse genetic system for PPV would be a useful tool for further studying the pathogenesis mechanisms of PPV, developing labeled vaccine and screening antiviral drug.

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.

Porcine Parvovirus Infection Causes Pig Placenta Tissue Damage Involving Nonstructural Protein 1 (NS1)-Induced Intrinsic ROS/Mitochondria-Mediated Apoptosis.

Porcine parvovirus (PPV) is an important pathogen causing reproductive failure in pigs. PPV-induced cell apoptosis has been recently identified as being involved in PPV-induced placental tissue damages resulting in reproductive failure. However, the molecular mechanism was not fully elucidated. Here we demonstrate that PPV nonstructural protein 1 (NS1) can induce host cell apoptosis and death, thereby indicating the NS1 may play a crucial role in PPV-induced placental tissue damages and reproductive failure. We have found that NS1-induced apoptosis was significantly inhibited by caspase 9 inhibitor, but not caspase 8 inhibitor, and transfection of NS1 gene into PK-15 cells significantly inhibited mitochondria-associated antiapoptotic molecules Bcl-2 and Mcl-1 expressions and enhanced proapoptotic molecules Bax, P21, and P53 expressions, suggesting that NS1-induced apoptosis is mainly through the mitochondria-mediated intrinsic apoptosis pathway. We also found that both PPV infection and NS1 vector transfection could cause host DNA damage resulting in cell cycle arrest at the G1 and G2 phases, trigger mitochondrial ROS accumulation resulting in mitochondria damage, and therefore, induce the host cell apoptosis. This study provides a molecular basis for elucidating PPV-induced cell apoptosis and reproductive failure.

Biology of Porcine Parvovirus (Ungulate parvovirus 1).

Porcine parvovirus (PPV) is among the most important infectious agents causing infertility in pigs. Until recently, it was thought that the virus had low genetic variance, and that prevention of its harmful effect on pig fertility could be well-controlled by vaccination. However, at the beginning of the third millennium, field observations raised concerns about the effectiveness of the available vaccines against newly emerging strains. Subsequent investigations radically changed our view on the evolution and immunology of PPV, revealing that the virus is much more diverse than it was earlier anticipated, and that some of the "new" highly virulent isolates cannot be neutralized effectively by antisera raised against "old" PPV vaccine strains. These findings revitalized PPV research that led to significant advancements in the understanding of early and late viral processes during PPV infection. Our review summarizes the recent results of PPV research and aims to give a comprehensive update on the present understanding of PPV biology.

The pathogenic role of torque teno sus virus 1 and 2 and their correlations with various viral pathogens and host immunocytes in wasting pigs.

The pathogenic role of torque teno sus virus (TTSuV) in swine is controversial among different studies. The present study intended to evaluate the potential pathogenicity of TTSuV based on its correlations with the histopathological changes, various common concurrently infected viral pathogens including porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PPV), as well as changes in the distribution and population of host immunocytes such as B lymphocytes, T lymphocytes, and macrophages by using the superficial inguinal lymph nodes (siLNs) of wasting pigs. A tissue microarray consisting of 270 available siLNs collected from 262 clinically wasting and 8 healthy pigs, respectively, were used for the detection of TTSuV1, TTSuV2, PCV2, PRRSV, and PPV by either in situ hybridization (ISH) or immunohistochemical (IHC) staining, and for the detection of various subsets of immunocytes by IHC staining with monoclonal antibodies to CD3, CD79a, and lysozyme. The slides were then subject to digital scanning followed by a semi-quantitative positive pixel evaluation for further statistical analysis. Although a high prevalence of TTSuV1 and/or TTSuV2 infection was noted in both wasting and healthy pigs, the wasting pigs had a significantly higher intensity in both TTSuV1 and TTSuV2 ISH-positive signals than healthy ones did. In the wasting pigs, a significant positive correlation in the tissue viral load was noted between TTSuV1 and TTSuV2 and between TTSuV2 and PCV2, but not between TTSuV1 and PCV2. Conversely, a significant negative correlation in the tissue viral load was revealed between TTSuV2, but not TTSuV1, and PRRSV. The tissue viral load of TTSuV1 was significantly correlated with B cell hyperplasia, while the tissue viral load of TTSuV2 was significantly correlated with increased macrophage population. The ISH positivity of TTSuV2 was significantly correlated with lymphoid depletion and granulomatous inflammation, which are the characteristic histopathological findings in postweaning multisystemic wasting syndrome-affected pigs. These findings suggest that both TTSuV species may have the potential involving the development of porcine circovirus-associated lymphoid lesions via alternating the host immune system.

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 1.39 g/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 60 kDa 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.

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine.

BACKGROUND: Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. METHODS: A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. RESULTS: Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. CONCLUSIONS: In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

Genetic elements in the VP region of porcine parvovirus are critical to replication efficiency in cell culture.

Factors controlling porcine parvovirus (PPV) replication efficiency are poorly characterized. Two prototype strains of PPV, NADL-2 and Kresse, differ greatly in pathogenic capacity both in vivo and in vitro, yet their genomic sequence is nearly identical (13 single-nucleotide substitutions and a 127-nucleotide noncoding repeated sequence). We have created a series of chimeras of these strains to identify the genetic elements involved in replication efficiency in the host porcine cell line. While the capsid proteins ultimately determine viral replication fitness, interaction between the NS1 protein and the VP gene occurs and involves interaction with the noncoding repeated sequence.

Real-time PCR to detect and analyze virulent PPV loads in artificially challenged sows and their fetuses.

To establish a real-time polymerase chain reaction with SYBR Green for detection and quantification of porcine parvovirus (PPV) in porcine tissues, two primers specific for the non-structural protein 1 gene were designed. The detection limit of this assay was 3-23 gene copies/reaction, equivalent to 0.001 TCID(50)/ml. The assay was linear over a 10(6) dilution range of template concentrations. Other porcine pathogens involved in reproductive disorders (porcine circovirus 2, porcine reproductive and respiratory virus, pseudorabies virus, classical swine fever virus) were negative by this assay. This assay could detect PPV titres at least 10(5) smaller than the hemagglutination assay. To better understand the pathogenesis of PPV, the levels of viral DNA in various tissues of artificially challenged sows and their fetuses were quantified with this method. The virus was found mainly in the heart, lung, spleen, kidney, and endometrium of sows, and mainly in the heart, spleen, lung, and testis of fetuses. This study provides a new tool for the study of PPV infection and distribution in sows and their fetuses.

A laboratory study of survival of selected microorganisms after heat treatment of biowaste used in biogas plants.

The aim of the study was to assess the effect of pasteurisation, as set by the European regulation EC 1774/2002, on selected pathogens and indicator organisms. Unpasteurised substrate (biowaste), including animal by-products from a full-scale biogas plant was heat treated under laboratory conditions at 70 degrees C and 55 degrees C for 30 min and 60 min. Heat treatment at 55 degrees C for 60 min was not sufficient to achieve a hygienically acceptable product. Heat treatment at 70 degrees C for 30 min and 60 min was effective in reducing pathogenic bacteria, Ascaris suum eggs, Swine vesicular disease virus and indicator organisms. However, this level of pasteurisation will still not reduce the quantity of Clostridia spores, or completely inactivate heat-resistant viruses such as Porcine parvovirus or Salmonella phage 28B. The results still give cause for some concern regarding the use of digested residue from biogasplants in agriculture.

Study of the virulence and cross-neutralization capability of recent porcine parvovirus field isolates and vaccine viruses in experimentally infected pregnant gilts.

The pathogenicity of two recent German field isolates of Porcine parvovirus (PPV-27a and PPV-143a) and two vaccine viruses [PPV-NADL-2 and PPV-IDT (MSV)], which are used for the production of inactivated vaccines, was investigated by inoculation of pregnant sows at day 40 of gestation. Post-infection sera of these sows as well as antisera prepared in rabbits by immunization with the four above-mentioned PPV isolates and with the virulent strain PPV-Challenge (Engl.) were tested for their homologous and heterologous neutralization activities. All antisera had high neutralization activity against the vaccine viruses, the PPV-Challenge (Engl.) virus and PPV-143a, but much lower activity against PPV-27a. These results suggest that PPV-27a represents a new antigenic variant or type of PPV and vaccines based on the established vaccine viruses may not be fully protective against this field isolate. PPV-27a has been characterized based on the amino acid sequences of the capsid protein as a member of a new and distinct PPV cluster (Zimmermann et al., 2006). Interestingly, the homologous neutralizing antibody titres of the sera of all three pigs and both rabbits inoculated or immunized with PPV-27a were 100- to 1000-fold lower than the heterologous titres against any of the other viruses. The low homologous neutralizing antibody titres suggest a possible, yet undefined, immune escape mechanism of this PPV isolate.

A colorimetric assay for viral agents that produce cytopathic effects.

Many animal viruses produce cytopathic effects in their host cells during a productive infection. While some virus infections can be assayed by the production of plaques, many viruses, while producing cytotoxicity, do not easily form plaques, or do not form plaques at all. Additionally, viruses within families such as the parvoviruses may have different preferred forms of titration making comparative virology difficult even among related groups. Porcine parvovirus (PPV), canine parvovirus (CPV), and minute virus of mice (MVM) are usually titrated using different infectivity assays. A direct comparison of infectious virus titer between these parvoviruses was sought, and a tetrazolium salt assay, MTT has been applied to measure cytopathic effect produced by viral infection for different members of the parvovirus family. Infectious PPV measured using the MTT and the TCID50 assays exhibited excellent correlation and titers for CPV and MVM were consistently duplicated using the MTT assay. The MTT assay was also applied to an unrelated virus, Sindbis, which is routinely titrated by plaque assay. MTT titration of Sindbis virus mutants was found to be valuable for preliminary screening. This assay can be adapted, by correlation to an accepted titration method, to any viral system which produces measurable cytopathic effect.

Coinfecção experimental de circovírus suíno tipo 2 isolado no Brasil e parvovírus suíno em suínos SPF / Experimental coinfection of SPF pigs with porcine circovirus type 2 isolated in Brazil and porcine parvovirus

Avaliou-se a patogenicidade do circovírus suíno tipo 2 (PCV2) isolado no estado de Santa Catarina mediante coinfecção experimental com parvovírus suíno (PPV). Foram utilizados 24 leitões specific pathogen free (SPF) com cinco dias de idade, distribuídos em quatro grupos (G), alojados em salas independentes e inoculados por via intranasal: G1 - controle (n=4); G2 - inoculados com PCV2 (n=7); G3 - inoculados com PPV (n=6); G4 - inoculados com PCV2 e PPV (n=7). Os animais foram monitorados diariamente para avaliação clínica e necropsiados 48 dias após a infecção. As principais lesões anatomopatológicas observadas nos suínos do G2 e G4 foram: aumento do volume dos linfonodos, depleção linfocitária com redução dos folículos linfóides nos órgãos linfocitários e presença de infiltrado eosinofílico nos linfonodos. A técnica de nested-PCR para PCV2 foi utilizada detectando DNA viral em órgãos de todos os animais do G2 e G4. O PCV2 infectou suínos SPF por via intranasal e foi detectado em outros órgãos, com mais lesões histopatológicas e em maior proporção nos animais coinfectados com PPV (G4), quando comparados aos infectados somente com PCV2 (G2).

The virulence of porcine circovirus type 2 (PCV2) isolated in Santa Catarina State by coinfection with porcine parvovirus (PPV) was investigated. Twenty-four, 5-day-old SPF pigs were distributed into four groups, housed in separate rooms and inoculated by intranasal route: G1 - control (n=4); G2 - inoculated with PCV2 (n=7); G3 - inoculated with PPV (n=6); G4 - inoculated with PCV2 and PPV (n=7). The animals were monitored daily for clinical evaluation and were necropsied 48 days after the infection. The pathological lesions seen in G2 and G4 pigs were: enlargement of lymph nodes, mild to moderate lymphoid cell depletion, affecting lymphoid follicles in lymphoid organs and presence of infiltration by eosinophils in lymph nodes. PCV2 DNA was detected by a nested-PCR in all pigs of G2 and G4. These findings confirmed that pigs were successfully infected intranasally with PCV2. The presence of PCV2 DNA in tissue samples and the pathological lesions were more evident in pigs infected with both PCV2 and PPV than in pigs infected with PCV2 alone.

Tissue distribution of two field isolates and two vaccine strains of porcine parvovirus in foetal organs after experimental infection of pregnant sows as determined by real-time PCR.

The aim of this study was to investigate the tissue distribution of two different field isolates and two vaccine strains of porcine parvoviruses (PPV) in infected piglets after transplacental infection. The viral load in 10 different foetal organs was determined by real-time polymerase chain reaction assays with SYBR Green targeting the viral VP2 gene and the genomic c-myc gene in 12 foetuses. The viral load in foetal tissues differed greatly among the different parvoviruses. Between one virulent field isolate compared with the other field isolate and the vaccine strains, the detected viral copy number differed in an order of magnitude of 10(9). The virulent isolate contained PPV in all 10 organs with viral loads varying between 10(11) and 10(15) per 10(6) cells. Concerning the other field isolate and the two vaccine strains, if PPV was detected, in most of the cases the highest viral load was found in foetal kidneys with a maximum viral load of 10(3) per 10(6) cells. Additionally, PPV was found in the heart of one foetus, in the liver and duodenum of one foetus and in the thymus of one foetus with viral loads varying between 10(2.1) and 10(3.5) per 10(6) cells. In completely mummified foetuses with no discriminable organs of foetuses infected with the vaccine strains and the less virulent isolate, PPV was present in very low amounts or even below the detection limit.

Pathogenesis of postweaning multisystemic wasting syndrome reproduced by co-infection with Korean isolates of porcine circovirus 2 and porcine parvovirus.

Thirty-two colostrum-deprived conventional pigs aged 28 days were co-infected with Korean isolates of porcine circovirus 2 (PCV2) and porcine parvovirus (PPV) and observed for up to 35 days post-inoculation (dpi). Histopathologically, granulomatous inflammation, with or without intracytoplasmic inclusion bodies, was present in lymphoid tissues (e.g., lymph nodes, thymus, spleen and tonsil) from 20 dpi, being most severe at 24 dpi. PCV2 and PPV DNA were detected in the lymph nodes from 3 to 35 dpi by in-situ hybridization, but the labelling for both viruses was particularly intense and widespread at 20 and 24 dpi. A close relationship between the cells labelled for PCV2 and those labelled for PPV was revealed by examination of serial sections from lymph nodes, and PCV2 and PPV were also detected in peripheral blood monocytes from 3 to 35 dpi. Other tissues and cells in which PCV2 and PPV DNA were detected included macrophages in the tonsil, thymus, spleen, lung, liver, kidney and heart. Significantly more PCV2-positive cells than PPV-positive cells were detected in the lymph nodes at 5 to 35 dpi. The pathogenesis of postweaning multisystemic wasting syndrome (PMWS) reproduced in this study may be suggested thus: initial viral entry through tonsillar macrophages, followed within 3 days by viraemia; PCV2 and PPV replicate, at least to some extent, in circulating peripheral monocytes, contributing to the cell-associated viraemia and to viral distribution throughout the lymphoid tissues.

Effective use of gamma irradiation for pathogen inactivation of monoclonal antibody preparations.

Gamma irradiation has been used for decades as an effective method of pathogen inactivation of relatively inert materials. Until recently, its application to biologicals has resulted in unacceptable losses in functional activity. In this report we demonstrate that the damaging secondary effects of gamma irradiation can be controlled while maintaining the pathogen inactivation properties due to damage by primary effects. Control is achieved by a combination of protection from free radical damage to a monoclonal antibody through the use of the antioxidant ascorbate and by freeze-drying to minimize the potential for generating free radicals. The data demonstrate a synergy of these two approaches that results in quantitative recovery of functional activity while maintaining the ability to inactivate greater than 5 logs of porcine parvovirus infectivity.

Simultaneous detection of porcine circovirus 2 and porcine parvovirus in naturally and experimentally coinfected pigs by double in situ hybridization.

A technique for double in situ hybridization to simultaneously detect porcine circovirus 2 (PCV2) and porcine parvovirus (PPV) in the same tissue section was developed and applied to lymph node and spleen from 8 pigs experimentally coinfected with PCV2 and PPV and 20 pigs with naturally occurring postweaning multisystemic wasting syndrome. For double labeling studies, the tissue samples were processed sequentially, first for PPV in situ hybridization using a digoxigenin-labeled probe and then for PCV2 in situ hybridization using a biotinylated probe. Positive cells contained reaction products for PCV2 and PPV, respectively. Both PCV2 DNA and PPV DNA were observed mainly in the cytoplasm but occasionally in the nucleus. With double in situ hybridization, both PCV2 DNA and PPV DNA were simultaneously detected in lymph node and spleen. This double labeling technique for the detection of PCV2 and PPV is suitable both for pathogenesis studies and for diagnostic applications.