A novel strain of porcine adenovirus detected in urinary bladder urothelial cell culture

Abstract

Contamination of cell cultures is the most common problem encountered in cell culture laboratories. Besides the secondary cell contaminations often occurring in the cell laboratories, the contaminations originating from donor animal or human tissue are equally as common, but usually harder to recognize and as such require special attention. The present study describes the detection of porcine adenovirus (PAdV), strain PAdV-SVN1 in cultures of normal porcine urothelial (NPU) cells isolated from urinary bladders of domestic pigs. NPU cell cultures were evaluated by light microscopy (LM), polymerase chain reaction (PCR), and additionally assessed by transmission electron microscopy (TEM). Characteristic ultrastructure of virions revealed the infection with adenovirus. The adenoviral contamination was further identified by the sequence analysis, which showed the highest similarity to recently described PAdV strain PAdV-WI. Additionally, the cell ultrastructural analysis confirmed the life-cycle characteristic for adenoviruses. To closely mimic the in vivo situation, the majority of research on in vitro models uses cell cultures isolated from human or animal tissue and their subsequent passages. Since the donor tissue could be a potential source of contamination, the microbiological screening of the excised tissue and harvested cell cultures is highly recommended.

Figure 1

Micrographs of normal porcine urothelial (NPU) cell cultures of the XIII passage on porous membranes on a first (A) and third (B) day after seeding at a density of 2 × 10⁵ viable cells/cm². Note the fast cell attachment on the first day, where NPU cells are almost confluent, and an increased cell desquamation on a third day after seeding, where numerous rounded and detached NPU cells can be seen (arrows). Scale bars: 100 µm.

Figure 2

Transmission electron micrograph of negatively stained adenovirus from NPU cell culture suspension. The size and appearance of the viral particles are characteristics for the Adenoviridae family. Scale bar: 50 nm.

Figure 3

Gel electrophoresis showing 632 bp polymerase chain reaction (PCR) products of the amplified hexon gene fragment. Lane 1: molecular mass marker (DNA ladder 100 bp plus); lane 2: negative control; lane 3: passage XI of NPU cell culture; lane 4: passage XI of NPU cell culture, 10-fold dilution; lane 5: passage XIII of NPU cell culture; lane 6: passage XIII of NPU cell culture, 10-fold dilution; lane 7: passage X of NPU cell culture; lane 8: passage X of NPU cell culture, 10-fold dilution; lane 9: positive control (HAdV DNA).

Figure 4

Phylogenetic tree of partial polymerase (A) and hexon (B) deduced amino acid sequences. Neighbor-Joining phylogenetic trees were constructed with bootstrap test of 1000 replicates, showing percentages next to the branches (values below 50 are not shown). The representative strains from GenBank: HAdV-3, DQ086466; HAdV-4, AY487947; HAdV-5, AC_000008; HAdV-12, X73487; HAdV-40, L19443; HAdV-54, AB333801; SAdV-1, AY771780; SAdV-3, AY598782; TSAdV-1, AC_000190; BAdV-1, NC_006324; BAdV-2, AC_000001; BAdV-3, AC_000002; BAdV-4, AF036092; PAdV-3, AF083132; PAdV-5, AF289262; PAdV-WI, JF699045; BtAdV-3, GU226970; BtAdV-2, JN252129; CAdV-1, AC_000003; CAdV-2, AC_000020; RAdV-1, EU715130; FAdV-1, AC_000014. HAdV—human adenovirus, SAdV—simian adenovirus, TSAdV—tree shrew adenovirus, BAdV—bovine adenovirus, PAdV—porcine adenovirus, BtAdV—bat adenovirus, CAdV—canine adenovirus, RAdV—raptor adenovirus, FAdV—fowl adenovirus.

Figure 5

Transmission electron micrographs of PAdV-SVN1 in NPU cells isolated from porcine urinary bladder. PAdVs enter the NPU cells by endocytotic uptake (arrow on A´). After the endocytosis, the virions are found in late endosomes (A) or sporadically scattered in the cytosol (arrows on B). After the cell internalization, the virions are found in the nucleus, organized in larger paracrystalline arrays or spread throughout the nucleus in smaller clusters (B). Commonly the condensed chromatin at the nuclear envelope of infected NPU cells is noted (asterisks on B and C). The possible mechanism of nuclear escape includes the separation of nuclear envelope and merger of the nucleus full of virions with the cytosol (black arrowheads on C indicate the nuclear envelope, white ones the plasma membrane), or maybe even the individual release of virions through the nuclear pore (arrow on D). After re-entry to the cytosol, the virions are found both, in the large paracrystalline arrays (E) or randomly distributed throughout the cytosol (F), with frequently observed degradation of the cytoplasm at the site of their presence (F). At this point, the initiation of NPU cell necrosis can be observed. With the progress of cell lysis, the cell detaches from the epithelium, i.e., urothelium (G); the nucleus disintegrates and condensed chromatin is moved near the plasma membrane (asterisks on G). At the final stage of necrosis, virions leave the cell through the lysed plasma membrane (arrows on H). Scale bars: (A, C and E) 1 µm; (B) 20 µm; (D and H) 200 nm; (F) 500 nm, (G) 2 µm.