1 Centre de Recerca en Sanitat Animal (CReSA) Departament de Sanitat i d'Anatomia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
2 Fort Dodge Veterinaria SA, R&D Department, 17813, Vall de Bianya, Girona, Spain
3 P11 (Neuartige Viren/Xenotransplantation), Robert Koch-Institut, 13353, Berlin, Germany
Correspondence
M. Roca
merce.roca{at}uab.es
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ABSTRACT |
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INTRODUCTION |
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PMWS usually affects 2- to 3·5-month-old pigs, although the disease has been described in a wider age range, between 3 days and 6 months (Hirai et al., 2001; Segales & Domingo, 2002
). The disease is characterized clinically by growth retardation, paleness of the skin, dyspnoea and enlargement of inguinal lymph nodes; occasionally, jaundice and diarrhoea are observed (Harding & Clark, 1997
; Rosell et al., 1999
). At necropsy, the most frequent lesions are enlargement of lymph nodes and non-collapsed, tan-mottled lungs (Rosell et al., 1999
). The main histological lesions of PMWS consist of a variable degree of lymphocyte depletion with loss of follicles together with histiocytic and multinucleate giant cell infiltration in the lymphoid tissues, and lymphohistiocytic inflammatory infiltrations in a wide range of tissues (Clark, 1997
; Rosell et al., 1999
). Although PCV2 has been systematically associated with PMWS microscopic lesions, the full clinicopathological spectrum of the disease has been traditionally difficult to reproduce using experimental models with PCV2 only (Allan et al., 1999
; Balasch et al., 1999
; Kennedy et al., 2000
; Magar et al., 2000
). These results, and the fact that concomitant viral infections (Allan et al., 1999
; Harms et al., 2001
; Krakowka et al., 2000
; Rovira et al., 2002
) or non-infectious immunostimulation (Krakowka et al., 2001
) seem to trigger the clinical disease, suggest that PMWS is a multifactorial disease where PCV2 is strictly necessary but not a sufficient factor to develop the clinical outcome observed under field conditions (Allan et al., 2000a
, b
; Ellis et al., 1999
; Krakowka et al., 2000
; Rovira et al., 2002
). However, it must be remarked that some experimental studies have reproduced the disease in a significant number of animals using, apparently, only PCV2 as the inoculum (Allan et al., 2002
; Bolin et al., 2001
; Harms et al., 2001
).
The virus inocula used in several trials were obtained from tissue homogenates corresponding to PMWS-affected pigs or PCV2 isolated and propagated in cultured cells. These sources of virus may contain other common swine agents, as has been observed in at least one experiment (Ellis et al., 1999). Therefore, it is possible that the virus stocks used in these experiments were not pure and the reproduced disease and/or pathological lesions may not be attributable to PCV2 infection only (Fenaux et al., 2002
). This problem should be resolved with the use of infectious DNA clones of a pure source of PCV2. The availability of infectious clones offers an opportunity for analysis and modification of viral genomes at the molecular level and has greatly aided research on virus replication, pathogenesis and vaccine development (Boyer & Haenni, 1994
). In fact, a previous study used a molecular DNA clone of a USA strain of PCV2 injected directly into the liver and lymph nodes; this work was carried out to demonstrate that a biologically pure and homogeneous infectious virus stock could be used for PCV2 pathogenesis studies. In this experiment, mild histopathological PMWS characteristic lesions were reproduced (Fenaux et al., 2002
). Furthermore, chimeric infectious DNA clones of PCV2 have been developed, demonstrating that the one containing ORF2 capsid gene of pathogenic PCV2 cloned into the non-pathogenic PCV1 genomic backbone induces specific antibody response to the pathogenic PCV2 capsid antigen, but is attenuated in pigs (Fenaux et al., 2003
).
These latter studies (Fenaux et al., 2002, 2003
) used a USA PCV2 strain for the construction of the chimeric and non-chimeric DNA infectious clones. Although differences in pathogenicity among PCV2 strains recovered from PMWS cases have not been clearly established, certain differences in genotype by geographical regions have been indicated (Larochelle et al., 2002
; Mankertz et al., 2000
). The purpose of this study is to describe the generation of an infectious clone of a European strain of PCV2 and its testing under in vitro and in vivo conditions. For the latter purpose, the infectious PCV2 DNA clone was inoculated in conventional pigs by intramuscular and intraperitoneal routes.
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METHODS |
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Monitoring of the recombination event by PCR.
Since expression of viral antigen could originate from plasmid pIC-PCV2 as well as from the recombinant virus genome, the presence of pIC-PCV2 and PCV2 as a product of homologous recombination was monitored by PCR. For this purpose, DNA was isolated from cells 7 days after transfection and amplified with primers F199 (5'-CCACATCGAGAAAGCGAAAGGAAC-3') and B413 (5'-TCCGTGGACTGTTCTGTAGC-3') to distinguish between the input DNA and the recombination product.
Southern hybridization.
Low molecular mass DNA was isolated from cells, separated on a 1 % agarose gel, transferred onto a nylon membrane and visualized by Southern hybridization as described earlier (Mankertz et al., 1997). A PCV2-specific DIG-labelled probe of 366 bp (nt 37402, cap gene) was used, which was generated by PCR. For this purpose, a DIG-labelling PCR reaction (Roche Molecular Biochemicals) was set up with plasmid pSVL-cap(PCV2) as the template and using primers F243 (5'-TTAGGGTTTAAGTGGGGGGTCTTTA-3') and B244 (5'-CAGGGTGACAGGGGAGTGGG-3') (Mankertz & Hillenbrand, 2001
).
Animals.
Eighteen pigs from three different litters, selected for a low serological PCV2 titre in sows, were used. The piglets were weaned at 2 weeks of age, bled, ear tagged and kept in isolated experimental facilities. All pigs were found to have low titres of antibodies to PCV2 (1 : 20 to 1 : 320) by an immunoperoxidase monolayer assay (IPMA) (Rodriguez-Arrioja et al., 2000) when between the age of 30 and 40 days (day 0 of the experiment). The pigs were seronegative to porcine reproductive and respiratory virus (PRRSV), pseudorabies virus (PRV), porcine parvovirus (PPV), swine influenza virus (SIV), transmissible gastroenteritis coronavirus (TGEV), porcine respiratory coronavirus (PRCV), and Actinobacillus pleuropneumoniae, Pasteurella multocida, Bordetella bronchiseptica and swine erysipelas. Also at day 0 of experiment, serum, and nasal and rectal swabs were taken to be analysed by a PCV2 PCR described previously (Quintana et al., 2002
). All the pigs were found to be negative for PCV2.
PCV2 inoculum.
Swine kidney (SK) cells free of PCV1 were inoculated with the Spanish PCV2 isolate SPA3 (GenBank reference AF201310), which came from a lymphoid tissue homogenate of a PMWS-affected pig. This homogenate had been previously tested by PCR and/or IPMA to exclude the presence of PRRSV, PPV, PRV, SIV, TGEV and PCV1. After three serial passages of SK cells, the supernatant was recovered, clarified by centrifugation at 650 g, and further concentrated by ultrafiltration. The viral pellet was resuspended in Dulbecco's modified Eagle's medium, titrated with non-infected SK cells (105·4 TCID50 ml1) aliquoted, and frozen at 80 °C until used.
In vivo experimental design.
Pigs were distributed randomly into three groups and kept in different isolation rooms. On day 0 of the experiment, the first group of six pigs was inoculated intranasally (i.n.) with 5 ml of PCV2 inoculum (106 TCID50 per pig). The second group of six animals was inoculated intramuscularly (i.m.) with 2 ml of PCV2 infectious clone (300 µg of pIC-PCV2 per pig). Finally, the last group of six pigs was inoculated intraperitoneally (i.p.) with 2 ml of the infectious clone (300 µg of pIC-PCV2 per pig). Pigs were clinically monitored daily, weighed weekly, rectal temperature was measured three times a week and blood samples were collected weekly. Pigs were euthanized on day 35 post-inoculation (p.i.) by an overdose of intravenous sodium thiobarbital, in accordance with the European Guidelines for Animal Welfare. A complete necropsy was performed of all pigs, and lung, inguinal superficial lymph nodes, tonsil, thymus, spleen, liver, kidney, ileum and bone marrow samples were collected and fixed by immersion in 10 % buffered formalin.
Pathological studies.
Pig tissues were subsequently dehydrated through graded alcohols and embedded in paraffin wax, sectioned at 4 µm thick and stained with haematoxylin and eosin (HE). Moreover, serial sections were mounted on silane-treated slides and an in situ hybridization (ISH) technique to detect PCV2 nucleic acid using a specific 40 nt probe was performed as described previously (Rosell et al., 1999, 2000
).
PCV2 antibody detection.
IPMA described previously (Rodriguez-Arrioja et al., 2000) was performed to detect antibodies to PCV2 in serum samples obtained weekly (0, 7, 14, 21, 28 and 35 days p.i.).
Polymerase chain reaction (PCR) to detect PCV2 genome.
DNA was extracted from 200 µl of serum sample using Nucleo Spin Blood (Macherey-Nagel) according to the manufacturer's instructions. The PCR protocol used has been described previously (Quintana et al., 2002). The amplified products were run using a 2 % agarose gel and visualized by staining with 0·5 µg ethidium bromide ml1. The length of the amplicon for this primer pair was 656 bp.
Sequence analysis, sequence alignments and phylogenetic analysis.
Positive PCV2 PCR serum samples corresponding to one pig of each inoculated group PCV2 i.n., pIC-PCV2 i.m. and pIC-PCV2 i.p. were selected to complete the sequence and confirm that it corresponded to the original sequences of the viruses (SPA3 and GER3). The extracted products were amplified with a variety of sequencing primers (data not shown). To extract and purify DNA from the agarose gel, the MinElute gel extraction kit (Qiagen) was used according to the manufacturer's protocol. Finally, computer analysis and alignment were done using the CLUSTALW multiple alignment function of the BIOEDIT program (version 5.0.9, North Carolina State University). Phylogenetic trees were constructed using the Neighbour-Joining program in the MEGA2 package (version 2.1) taking into account 1000 bootstraps.
TaqMan PCR for PCV2.
To quantify PCV2 DNA in serum samples at 35 days p.i., a real-time fluorescent-probe PCR was used (Olvera et al., 2004). The assay was performed in triplicate.
Statistical analyses.
Statistical analyses were performed using the SAS system (SAS/STAT Users' Guide: Statistics, version 8. SAS Institute). A mixed linear model (Verbeke & Molenberghs, 2000) was designed using the mixed procedure for the statistical analysis of the weight and rectal temperatures between different groups. The results of the ISH and the PCR were analysed by Fisher's Exact Test. Finally, the results of the IPMA were analysed by a negative binomial test using the Genmod procedure. A generalized linear model was used for the statistical analysis of the TaqMan PCR results.
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RESULTS |
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Finally, seroconversion was observed in all groups (Table 1). PCV2 i.n. inoculated pigs seroconverted as early as 14 days p.i., while the other groups started seroconverting on days 21 (pIC-PCV2 i.p. inoculated pigs) and 28 p.i. (pIC-PCV2 i.m. inoculated pigs). These differences were statistically significant among all groups (P<0·0001). At the end of the experimental period (35 days p.i.), all pigs inoculated with PCV2 i.n. and four out of six pIC-PCV2 i.p. inoculated pigs had high PCV2 antibody titres (ranging from 1 : 5120 to 1 : 20 480). On the other hand, the remaining two pIC-PCV2 i.p. and four out of six pIC-PCV2 i.m. inoculated pigs had low to intermediate titres (1 : 320 to 1 : 1280). Two pigs from the pIC-PCV2 i.m. inoculated group did not seroconvert.
Clinical and pathological evaluation of PCV2/pIC-PCV2 inoculated pigs
All pigs remained clinically healthy during the whole experimental period. No significant differences were observed in rectal temperatures between the three studied groups. Numerically, the weight increase over the experimental period was higher in the pIC-PCV2 i.p. inoculated group followed by the pIC-PCV2 i.m. inoculated group, and finally by the PCV2 i.n. inoculated group. However, significant differences (P<0·013) in body weight were observed only on day 35 p.i. between the PCV2 i.n. and pIC-PCV2 i.p. inoculated pigs. No significant differences in body weight were observed among the groups during the rest of the experimental period.
Macroscopically, three out of six PCV2 i.n. inoculated pigs had a slight increase in the size of the mesenteric and mediastinal lymph nodes. Only one out of six animals that received the pIC-PCV2 i.m. had a mild increase in the size of the mesenteric lymph node. Finally, two out of six pIC-PCV2 i.p. inoculated pigs had a mild increase in the size of the mesenteric lymph node. No other significant gross lesions were observed in the inoculated pigs.
Microscopic lesions are summarized in Table 2. Lesions observed in lymphoid tissues consisted of a mild lymphocyte depletion with moderate histiocytic infiltration; multinucleate giant cells were very occasionally seen in the lymphoid organs. Lung lesions consisted of a mild interstitial pneumonia. Also, one pig that received pIC-PCV2 i.m. had a mild multifocal lymphoplasmacytic hepatitis. Three animals from the PCV2 i.n. and one pig from the pIC-PCV2 i.p. inoculated groups had mild interstitial nephritis.
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DISCUSSION |
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The generated infectious clone contained an overlength genome of a German PCV2 isolate with a partial duplication that enabled the virus to recircularize by homologous recombination and jump out of the plasmid backbone to initiate a natural infection. When this plasmid was applied in vitro, 20 % of the cells transfected with pIC-PCV2 showed expression of viral antigen, as determined by IFA. It is known that if two identical or similar sequences are present within one DNA molecule, this molecule may undergo homologous recombination (Valerie & Povirk, 2003; West, 2003
). Therefore, to discriminate between expression from the original input DNA and the product of recombination, a monitoring PCR was used. This approach led to the identification of recombinant virus, while no input DNA was detected. Taken together, these results show that infection by pIC-PCV2 delivers genomic PCV2 DNA specifically into susceptible cells and leads to the expression of a functional virus genome.
In the experimental trial, all pigs that received the PCV2 infectious clone became infected. Pathological, serological and virological (detection and quantification of the virus) results of these animals were very similar to those obtained for the IN infected pigs with a PCV2 strain propagated in cell culture. However, none of the pigs from this study developed clinical PMWS, a condition where PCV2 is considered the major infectious agent involved (Allan et al., 1999; Bolin et al., 2001
; Krakowka et al., 2001
), although it is not necessarily sufficient to trigger the clinical disease (Segales & Domingo, 2002
). Only mild pathological lesions characteristic of PMWS such as lymphoid depletion and granulomatous infiltration (Rosell et al., 1999
) were observed in lymphoid tissues. In fact, the present results are also very similar to published work which tested an infectious PCV2 clone based on an American strain of the virus (Fenaux et al., 2002
). In this latter report, the PCV2 genomic DNA clone was injected directly into the liver and superficial iliac lymph node of SPF pigs. These authors used those inoculation routes based on the fact that PCV2 replicates in the lymph nodes and liver during the natural (Choi & Chae, 2000
; Kiupel et al., 1999
; Rosell et al., 1999
, 2000
) and experimental (Allan et al., 1999
; Bolin et al., 2001
; Harms et al., 2001
; Krakowka et al., 2000
) infection. In the present study, the i.m. and i.p. routes of inoculation were chosen. The former one is a traditional and well described route for inoculation of DNA vaccines and plasmids (Ulmer et al., 1997
; van Drunen Littel-van den Hurk et al., 2000
; Wolff et al., 1990
), and it has been demonstrated to be an efficient route to generate both humoral and cellular immunity against several pathogens in different species, including swine (Cox et al., 1993
; Gerdts et al., 1997
; Haagmans et al., 1999
; van Rooij et al., 1998
). The intraperitoneal route, however, is not a usual route to inoculate DNA plasmids in pigs. This route was thought to be useful since the virus would be transported, either after phagocytosis by intraperitoneal macrophages or in suspension in the lymph, towards the cranial sternal lymph nodes of the ventral thoracic lymphocentrum, as has been demonstrated in other species (Marco et al., 1992
). Therefore, the intraperitoneal route would represent a very direct way to reach the lymph nodes of the pig and easier than direct inoculation into lymph nodes. In the present study, the intraperitoneal route was as effective or more than the intramuscular route, at least in terms of infection based on the serological, PCR and TaqMan PCR results. Moreover, the PCV2 genomic DNA clone did not show any difference in viral load at 35 days p.i. when compared to the i.n. route using a PCV2 isolate propagated in cell culture.
Certain differences were observed among groups regarding dynamics of seroconversion and viral detection in serum. Although almost all animals seroconverted by day 35 p.i., pigs that received the PCV2 strain propagated in cell culture seroconverted before groups that received the infectious clone. Furthermore, the pigs that received the DNA clone i.p. seroconverted before (and had higher serological titres at different days p.i.) the ones that received it i.m. These results may suggest that to generate humoral immunity the intraperitoneal route is more efficient than the intramuscular route; however, these results need to be confirmed in an experiment with a higher number of inoculated animals.
The low level of maternal antibodies in the inoculated pigs at the start of the experiment did not seem to have any confounding influence on the onset of viraemia or seroconversion. Similarly, in a previous experiment performed by our group, we also observed that pigs with a low level of maternal antibodies developed PCV2 infection and even PMWS (Rovira et al., 2002). However, we were not able to make a definitive assessment as to whether low levels of maternal antibodies could prevent infection or modify its outcome.
The results of the present study further confirm that cloned PCV2 genomic DNA is infectious in vitro and in vivo, and that it is able to cause PMWS-like lesions in i.p. and i.m. experimentally infected pigs, administration routes that have not been tested previously. The use of infectious DNA clones removes the need for infectious virus stocks in cell cultures, decreases the risk of cell culture contamination with other virus and allows the generation of a biologically pure infectious PCV2. Although it was not the purpose of the present study, the results obtained open the possibility to explore the potential use of this PCV2 infectious clone as a DNA vaccine to control PMWS and other PCV2-associated diseases. In fact, recent published information on the pathogenic and immunogenic characteristics of a chimeric infectious DNA clone (Fenaux et al., 2003) have shown the potential use of these constructs as genetically engineered live-attenuated vaccines.
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ACKNOWLEDGEMENTS |
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Received 21 November 2003;
accepted 21 January 2004.