1 Center for Molecular Medicine and Infectious Diseases, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0342, USA
2 Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
3 Genentech Inc., 1 DNA Way, South San Francisco, CA, USA
Correspondence
X.-J. Meng
xjmeng{at}vt.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the PCV1 and PCV2 sequences obtained in this study are AY699796 and AY699793, respectively.
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MAIN TEXT |
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Xenotransplantation with porcine organs, cells and other porcine-derived products has received considerable attention in the last few years and could potentially alleviate the problems associated with the shortage of human organ donors (Meng, 2003; Tucker et al., 2003
). However, inadvertent transmission of potentially zoonotic porcine viruses to humans is of major concern. Antibodies to PCV1 have been reportedly detected in many animal species, including humans (Tischer et al., 1986
), and PCV2 has been shown to infect BALB/c mice (Kiupel et al., 2001
). However, others have reported contradictory results regarding the ability of PCV to cause cross-species infections (Ellis et al., 2000
; Quintana et al., 2002
). Nevertheless, the use of PCV-contaminated porcine organs, cells and medical products, such as factor VIII, heparin, insulin and pepsin, in humans through xenotransplantation procedures has raised concerns for potential human infections. The objective of this study was to determine whether a porcine-derived commercial pepsin product was contaminated with PCVs and, if so, whether or not the contaminating PCVs were still infectious in vitro and in vivo.
Samples of two different porcine-derived pepsin lots (two lyophilized 100 g vials from each lot) from a commercial company were tested for the presence of PCV1 and PCV2 DNA by PCR. For each vial of pepsin, 10 mg lyophilized product was dissolved in 100 µl PBS. DNA was extracted from the dissolved pepsin with a QIAamp DNA mini kit (Qiagen) according to the protocols supplied by the manufacturer. The MCV1 (5'-GCTGAACTTTTGAAAGTGAGCGGG-3') and MCV2 (5'-TCACACAGTCTCAGTAGATCATCCCA-3') primer pair was used to detect PCV1 or PCV2 DNA in the pepsin by PCR, as described previously (Fenaux et al., 2000). To amplify and sequence the complete genomes of PCV1 and PCV2 from the contaminated pepsin product, 11 nested sets of primer pairs were designed, based on the published sequences of PCV1 and PCV2 (Fenaux et al., 2000
). PCR products of the expected size were purified and sequenced directly by using the PCR primers.
Of the two lots of pepsin that were tested in this study, one (both vials) was positive for both PCV1 and PCV2 DNA by PCR, and the other one was negative. The complete genomes of the pepsin-derived PCV1 and PCV2 were determined to be 1759 and 1768 bp in length, respectively. To determine the extent of sequence identity between the pepsin-derived PCV1 and PCV2 and other known PCVs, the complete genomic sequences of four PCV1 and 31 PCV2 isolates that were available in GenBank were compared with those of the pepsin-derived PCV1 and PCV2. Pepsin-derived PCV2 displayed 9599 % nucleotide sequence identity with the published PCV2 sequences. Similarly, the pepsin-derived PCV1 shared 9899 % nucleotide sequence identity with the published PCV1 sequences. The pepsin-derived PCV1 and PCV2 shared 76 % nucleotide sequence identity with each other. A phylogenetic tree was constructed on the basis of the complete genomic sequences of 37 isolates of PCV1 and PCV2 from different geographical regions with the aid of the PAUP program (David L. Swofford, Smithsonian Institution, Washington, DC, USA) (Fig. 1). Phylogenetic analysis showed that the pepsin-derived PCV2 was related closely to North American PCV2 isolates and formed a minor branch (Fig. 1
). Similarly, the pepsin-derived PCV1 clustered with other PCV1 isolates, but formed a distinct minor branch.
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We and others have demonstrated previously that naked PCV2 or PCV1 genomic DNA was infectious when injected intramuscularly into live animals (Fenaux et al., 2002, 2003
, 2004a
, b
; Roca et al., 2004
). Therefore, the contaminating PCV DNA in pepsin, although non-infectious in vitro, could still be infectious when injected into animals. To further investigate the infectivity of the contaminating pepsin, 16 specific pathogen-free (SPF) pigs of 5 weeks of age were assigned randomly to three groups. Prior to inoculation at 1 day post-inoculation (d.p.i.), serum samples from all pigs in groups 1, 2 and 3 tested negative for PCV1 and PCV2 DNA. To maximize inoculation efficiency, each pig was given 1/4 of the inoculum intramuscularly and 3/4 intranasally. The five pigs in group 1 were each inoculated with 4 ml PBS as negative controls, the six pigs in group 2 were each inoculated with 400 mg PCV-contaminated pepsin dissolved in 4 ml PBS and the five pigs in group 3 were each inoculated with 4x104·3 TCID50 wild-type PCV2 as positive controls.
All pigs were monitored for clinical signs of disease by a team of two people. Pigs were weighed on a weekly basis. Rectal temperatures and clinical respiratory scores, ranging from 0 to 6 (0, normal; 6, severe) (Halbur et al., 1995), were recorded every other day from 5 to 39 d.p.i. Clinical observations, including evidence of central nervous system disease, liver disease (icterus), musculoskeletal disease and changes in body condition, were recorded daily. There was no difference in weight gain or mean rectal temperatures among any of the groups. From 19 to 42 d.p.i., the mean clinical scores recorded for positive-control group 3 pigs became more severe (P<0·05) than those recorded for animals in groups 1 and 2 (data not shown).
All animals received a complete necropsy at 42 d.p.i. The necropsy team was blinded to the infection status of the pigs at necropsy. An estimated percentage of the lung with grossly visible pneumonia was recorded for each pig, based on a previously described scoring system (Halbur et al., 1995). Other lesions, such as enlargement of lymph nodes (ranging from 0 for normal to 3 for three times normal size), were scored separately (Fenaux et al., 2004a
, b
). We examined the pathological lesions that were induced by PCV2 infection only, as PCV1 is non-pathogenic (Allan et al., 1995
). The group 1 negative-control pigs had no evidence of gross lymph-node or lung lesions. Two of the six pepsin-inoculated group 2 pigs had moderate enlargement of lymph nodes. The lesions in these two pigs were not attributable to PCV2, as serological and virological evidence of PCV2 infection in these pigs was lacking. The PCV2-inoculated group 3 pigs all had mild to severe enlargement of lymph nodes. The mean gross lesion scores in the lymph nodes of group 1 and group 2 pigs were not significantly different from each other, but were significantly less severe than that of PCV2-inoculated group 3 positive-control pigs. Lungs were free of gross visible pneumonia in all three groups.
Sections for histopathological examination were taken from lungs (five sections), heart, lymph nodes (tracheobronchial), tonsil, thymus, liver, spleen, small intestine and kidney. The tissues were examined in a blinded fashion and given a score for severity of lung, lymph-node, tonsil, spleen and liver lesions (Halbur et al., 1995). Lung scores ranged from 0 (normal) to 3 (severe lymphohistiocytic interstitial pneumonia). Liver scores ranged from 0 (normal) to 3 (severe lymphohistiocytic hepatitis). Lymphoid-tissue scores were for an estimated amount of lymphoid depletion (LD) and histiocytic replacement (HR) of follicles, ranging from 0 (normal) to 3 (severe LD and HR of follicles). Mild lymphoplasmacytic and histiocytic bronchointerstitial pneumonia was observed in three of five group 1 pigs and in five of six pepsin-inoculated group 2 pigs (Table 1
). All PCV2-inoculated group 3 pigs had mild to moderate lymphoplasmacytic and histiocytic bronchointerstitial pneumonia. In the pepsin-inoculated group 2 pigs, one animal had very mild LD and HR of the lymph-node follicles. All PCV2-inoculated group 3 pigs had mild to moderate LD and HR in lymph nodes and spleen tissues (Table 1
).
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Xenozoonosis due to the inadvertent transmission of porcine viruses from pig xenografts to human transplant recipients and the potential subsequent transmission of the virus to others are of major concern in xenotransplantation (Meng, 2003; Tucker et al., 2003
). In this study, we found that one of the two lots of porcine-derived commercial pepsin that were tested was positive for both PCV1 and PCV2 DNA. However, we were unable to infect PK-15 cells with the contaminated pepsin, suggesting that the contaminated PCV1 and PCV2 viruses in pepsin were probably inactivated and lacked infectivity in vitro. SPF piglets that were inoculated experimentally with the contaminated pepsin did not become viraemic for PCV1 or PCV2, nor did they seroconvert to PCV2; PCV2 antigen was not detected in the lymphoid tissues of pepsin-inoculated pigs. Compared to the PCV2-inoculated positive-control group, the negative-control pigs and the pepsin-inoculated pigs had no significant gross or microscopic lesions characteristic of PCV2 infection. The lack of PCV infectivity of the contaminated pepsin in vitro and in vivo is probably due to the pepsin-manufacturing process, which effectively inactivates the PCVs and degrades their genomic DNA. However, other porcine-derived products, such as factor VIII from pig plasma, may contain high titres of infectious PCVs and, thus, the manufacturing process may not completely inactivate the viruses. Porcine parvovirus (Soucie et al., 2000
) and porcine endogenous retrovirus (Takefman et al., 2001
) were detected in porcine-derived factor VIII. Therefore, it is important to test other porcine-derived medical and research products, such as factor VIII, heparin and insulin, for potential contamination by PCVs.
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ACKNOWLEDGEMENTS |
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Received 8 July 2004;
accepted 27 July 2004.