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Productive infection of bovine papillomavirus type 2 in the urothelial cells of naturally occurring urinary bladder tumors in cattle and water buffaloes.

Roperto S, Russo V, Ozkul A, Corteggio A, Sepici-Dincel A, Catoi C, Esposito I, Riccardi MG, Urraro C, Lucà R, Ceccarelli DM, Longo M, Roperto F - PLoS ONE (2013)

Bottom Line: L1 DNA was amplified by PCR, cloned and sequenced and confirmed to be L1 DNA.Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the neoplastic urothelium.Electron microscopic investigations detected electron dense particles, the shape and size of which are consistent with submicroscopic features of viral particles, in nuclei of neoplastic urothelium.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Veterinaria e Produzioni Animali, Sezione Malattie Infettive, Università di Napoli Federico II, Naples, Italy. sante.roperto@unina.it

ABSTRACT

Background: Papillomaviruses (PVs) are highly epitheliotropic as they usually establish productive infections within squamous epithelia of the skin, the anogenital tract and the oral cavity. In this study, early (E) and late (L) protein expression of bovine papillomavirus type 2 (BPV-2) in the urothelium of the urinary bladder is described in cows and water buffaloes suffering from naturally occurring papillomavirus-associated urothelial bladder tumors.

Methods and findings: E5 protein, the major oncoprotein of the BPV-2, was detected in all tumors. L1 DNA was amplified by PCR, cloned and sequenced and confirmed to be L1 DNA. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection was detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the neoplastic urothelium. Finally, the early protein E2, required for viral DNA replication and known to be a pivotal factor for both productive and persistent infection, was detected by Western blot and immunohistochemically. Electron microscopic investigations detected electron dense particles, the shape and size of which are consistent with submicroscopic features of viral particles, in nuclei of neoplastic urothelium.

Conclusion: This study shows that both active and productive infections by BPV-2 in the urothelium of the bovine and bubaline urinary bladder can occur in vivo.

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Related in: MedlinePlus

PCR amplification of DNA samples.a) Lane M, molecular mass marker (HyperLadder II Bioline); lanes 1–2 urinary bladder samples from healthy cows without BPV-2 L1 DNA; lanes 3–7 bladder tumors samples from five of twenty cows showing BPV-2 L1 DNA; lane C+, positive control (cloned BPV-2 DNA); lane C-, negative control (no DNA added). The arrow indicates the position of the 164 bp BPV-2 L1 PCR product. b) Lane M, molecular mass marker (HyperLadder II Bioline); lanes 1–2 urinary bladder samples from healthy buffaloes without BPV-2 L1 DNA; lanes 3–7 bladder tumors samples from five of twenty-one buffaloes showing BPV-2 L1 DNA; lane C+, positive control (cloned BPV-2 DNA); lane C-, negative control (no DNA added). The arrow indicates the position of the 164 bp BPV-2 L1 PCR product. The lower part of the figure shows 100% homology between the sequence of the amplicons in lanes 3–7 and the sequence of BPV-2 L1 found in Italy (GenBank M20219).
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pone-0062227-g002: PCR amplification of DNA samples.a) Lane M, molecular mass marker (HyperLadder II Bioline); lanes 1–2 urinary bladder samples from healthy cows without BPV-2 L1 DNA; lanes 3–7 bladder tumors samples from five of twenty cows showing BPV-2 L1 DNA; lane C+, positive control (cloned BPV-2 DNA); lane C-, negative control (no DNA added). The arrow indicates the position of the 164 bp BPV-2 L1 PCR product. b) Lane M, molecular mass marker (HyperLadder II Bioline); lanes 1–2 urinary bladder samples from healthy buffaloes without BPV-2 L1 DNA; lanes 3–7 bladder tumors samples from five of twenty-one buffaloes showing BPV-2 L1 DNA; lane C+, positive control (cloned BPV-2 DNA); lane C-, negative control (no DNA added). The arrow indicates the position of the 164 bp BPV-2 L1 PCR product. The lower part of the figure shows 100% homology between the sequence of the amplicons in lanes 3–7 and the sequence of BPV-2 L1 found in Italy (GenBank M20219).

Mentions: Table 1 and Table 2 report histological diagnosis of urothelial tumors of the urinary bladder of cattle and buffaloes, respectively, performed according to recent morphological criteria [22]. In all tumor cases, the BPV-2 E5 oncoprotein was detected by immunoprecipitation (Figure 1). As it has been shown that BPV-2 plays a crucial role in bladder carcinogenesis of large ruminants [14], [19] and since the cattle and buffaloes affected with urothelial tumors, here reported, were from Italy and Turkey respectively, we analyzed the sequence of the L1 gene, the most conserved PV gene, to assess any variation in BPV-2 between the two countries. PCR analysis was performed in all bovine and bubaline samples. L1 DNA was amplified and a band of ∼55 kD was shown both in benign and malignant tumors (Figure 2). Amplicon sequencing detected a DNA fragment composed of 164 bp (Figure 2) showing an absolute homology (100%) with the known sequences of BPV-2 L1 DNA. There were no differences in L1 DNA sequence between cattle and buffaloes. GenBank accession number of our L1 sequence is M20219.


Productive infection of bovine papillomavirus type 2 in the urothelial cells of naturally occurring urinary bladder tumors in cattle and water buffaloes.

Roperto S, Russo V, Ozkul A, Corteggio A, Sepici-Dincel A, Catoi C, Esposito I, Riccardi MG, Urraro C, Lucà R, Ceccarelli DM, Longo M, Roperto F - PLoS ONE (2013)

PCR amplification of DNA samples.a) Lane M, molecular mass marker (HyperLadder II Bioline); lanes 1–2 urinary bladder samples from healthy cows without BPV-2 L1 DNA; lanes 3–7 bladder tumors samples from five of twenty cows showing BPV-2 L1 DNA; lane C+, positive control (cloned BPV-2 DNA); lane C-, negative control (no DNA added). The arrow indicates the position of the 164 bp BPV-2 L1 PCR product. b) Lane M, molecular mass marker (HyperLadder II Bioline); lanes 1–2 urinary bladder samples from healthy buffaloes without BPV-2 L1 DNA; lanes 3–7 bladder tumors samples from five of twenty-one buffaloes showing BPV-2 L1 DNA; lane C+, positive control (cloned BPV-2 DNA); lane C-, negative control (no DNA added). The arrow indicates the position of the 164 bp BPV-2 L1 PCR product. The lower part of the figure shows 100% homology between the sequence of the amplicons in lanes 3–7 and the sequence of BPV-2 L1 found in Italy (GenBank M20219).
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Related In: Results  -  Collection

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pone-0062227-g002: PCR amplification of DNA samples.a) Lane M, molecular mass marker (HyperLadder II Bioline); lanes 1–2 urinary bladder samples from healthy cows without BPV-2 L1 DNA; lanes 3–7 bladder tumors samples from five of twenty cows showing BPV-2 L1 DNA; lane C+, positive control (cloned BPV-2 DNA); lane C-, negative control (no DNA added). The arrow indicates the position of the 164 bp BPV-2 L1 PCR product. b) Lane M, molecular mass marker (HyperLadder II Bioline); lanes 1–2 urinary bladder samples from healthy buffaloes without BPV-2 L1 DNA; lanes 3–7 bladder tumors samples from five of twenty-one buffaloes showing BPV-2 L1 DNA; lane C+, positive control (cloned BPV-2 DNA); lane C-, negative control (no DNA added). The arrow indicates the position of the 164 bp BPV-2 L1 PCR product. The lower part of the figure shows 100% homology between the sequence of the amplicons in lanes 3–7 and the sequence of BPV-2 L1 found in Italy (GenBank M20219).
Mentions: Table 1 and Table 2 report histological diagnosis of urothelial tumors of the urinary bladder of cattle and buffaloes, respectively, performed according to recent morphological criteria [22]. In all tumor cases, the BPV-2 E5 oncoprotein was detected by immunoprecipitation (Figure 1). As it has been shown that BPV-2 plays a crucial role in bladder carcinogenesis of large ruminants [14], [19] and since the cattle and buffaloes affected with urothelial tumors, here reported, were from Italy and Turkey respectively, we analyzed the sequence of the L1 gene, the most conserved PV gene, to assess any variation in BPV-2 between the two countries. PCR analysis was performed in all bovine and bubaline samples. L1 DNA was amplified and a band of ∼55 kD was shown both in benign and malignant tumors (Figure 2). Amplicon sequencing detected a DNA fragment composed of 164 bp (Figure 2) showing an absolute homology (100%) with the known sequences of BPV-2 L1 DNA. There were no differences in L1 DNA sequence between cattle and buffaloes. GenBank accession number of our L1 sequence is M20219.

Bottom Line: L1 DNA was amplified by PCR, cloned and sequenced and confirmed to be L1 DNA.Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the neoplastic urothelium.Electron microscopic investigations detected electron dense particles, the shape and size of which are consistent with submicroscopic features of viral particles, in nuclei of neoplastic urothelium.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Veterinaria e Produzioni Animali, Sezione Malattie Infettive, Università di Napoli Federico II, Naples, Italy. sante.roperto@unina.it

ABSTRACT

Background: Papillomaviruses (PVs) are highly epitheliotropic as they usually establish productive infections within squamous epithelia of the skin, the anogenital tract and the oral cavity. In this study, early (E) and late (L) protein expression of bovine papillomavirus type 2 (BPV-2) in the urothelium of the urinary bladder is described in cows and water buffaloes suffering from naturally occurring papillomavirus-associated urothelial bladder tumors.

Methods and findings: E5 protein, the major oncoprotein of the BPV-2, was detected in all tumors. L1 DNA was amplified by PCR, cloned and sequenced and confirmed to be L1 DNA. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection was detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the neoplastic urothelium. Finally, the early protein E2, required for viral DNA replication and known to be a pivotal factor for both productive and persistent infection, was detected by Western blot and immunohistochemically. Electron microscopic investigations detected electron dense particles, the shape and size of which are consistent with submicroscopic features of viral particles, in nuclei of neoplastic urothelium.

Conclusion: This study shows that both active and productive infections by BPV-2 in the urothelium of the bovine and bubaline urinary bladder can occur in vivo.

Show MeSH
Related in: MedlinePlus