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Isolation and genetic characterization of human coronavirus NL63 in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells.

Lednicky JA, Waltzek TB, McGeehan E, Loeb JC, Hamilton SB, Luetke MC - Virol. J. (2013)

Bottom Line: Human coronavirus NL63 was isolated and identified by RT-PCR and sequencing, and its replication in a fresh batch of RPTEC and another type of primary human kidney cells was confirmed.Whereas we are unable to determine whether the original RPTEC were pre-infected prior to their separation from other kidney cells, or had gotten contaminated with HCoV-NL63 from an ill laboratory worker during their preparation for commercial sale, our findings are a reminder that human-derived biologicals should always be considered as potential sources of infectious agents.Importantly, HCoV-NL63 replicates to high titers in some primary human kidney cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Environmental and Global Health, College of Public Health and Health Professions, University of Florida, Box 100188, Gainesville, FL 32610-0188, USA. jlednicky@phhp.ufl.edu

ABSTRACT

Background: Cryopreserved primary human renal proximal tubule epithelial cells (RPTEC) were obtained from a commercial supplier for studies of Simian virus 40 (SV40). Within twelve hrs after cell cultures were initiated, cytoplasmic vacuoles appeared in many of the RPTEC. The RPTEC henceforth deteriorated rapidly. Since SV40 induces the formation of cytoplasmic vacuoles, this batch of RPTEC was rejected for the SV40 study. Nevertheless, we sought the likely cause(s) of the deterioration of the RPTEC as part of our technology development efforts.

Methods: Adventitious viruses in the RPTEC were isolated and/or detected and identified by isolation in various indicator cell lines, observation of cytopathology, an immunoflurorescence assay, electron microscopy, PCR, and sequencing.

Results: Cytomegalovirus (CMV) was detected in some RPTEC by cytology, an immunofluorescence assay, and PCR. Human Herpesvirus 6B was detected by PCR of DNA extracted from the RPTEC, but was not isolated. Human coronavirus NL63 was isolated and identified by RT-PCR and sequencing, and its replication in a fresh batch of RPTEC and another type of primary human kidney cells was confirmed.

Conclusions: At least 3 different adventitious viruses were present in the batch of contaminated RPTEC. Whereas we are unable to determine whether the original RPTEC were pre-infected prior to their separation from other kidney cells, or had gotten contaminated with HCoV-NL63 from an ill laboratory worker during their preparation for commercial sale, our findings are a reminder that human-derived biologicals should always be considered as potential sources of infectious agents. Importantly, HCoV-NL63 replicates to high titers in some primary human kidney cells.

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Appearance of early RPTEC culture and of WI-38 cells during bioactive agent release assay. [A] Vacuolated RPTEC cells, 24 hr culture, 400X. [B] Non-infected WI-38 cells demonstrating expected fibroblast shapes, 400X. [C] WI-38 cells 12 hrs post-exposure to spent BGM from a 24 hr RPTEC culture, 400X.
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Figure 1: Appearance of early RPTEC culture and of WI-38 cells during bioactive agent release assay. [A] Vacuolated RPTEC cells, 24 hr culture, 400X. [B] Non-infected WI-38 cells demonstrating expected fibroblast shapes, 400X. [C] WI-38 cells 12 hrs post-exposure to spent BGM from a 24 hr RPTEC culture, 400X.

Mentions: Vacuoles were still present 24 hrs post-seeding of the RPTEC (and after the RGM change at 12 hrs) (Figure 1A), but there were no signs of contamination by extracellular bacteria or fungi. The pH at 37°C of fresh BGM was approximately 7.36 (within normal range), and ammonia was not detected using a salicylate-based method (data not shown). These findings suggested neither incorrect pH nor presence of ammonia in BGM were causing vacuolation of the RPTEC.


Isolation and genetic characterization of human coronavirus NL63 in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells.

Lednicky JA, Waltzek TB, McGeehan E, Loeb JC, Hamilton SB, Luetke MC - Virol. J. (2013)

Appearance of early RPTEC culture and of WI-38 cells during bioactive agent release assay. [A] Vacuolated RPTEC cells, 24 hr culture, 400X. [B] Non-infected WI-38 cells demonstrating expected fibroblast shapes, 400X. [C] WI-38 cells 12 hrs post-exposure to spent BGM from a 24 hr RPTEC culture, 400X.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716658&req=5

Figure 1: Appearance of early RPTEC culture and of WI-38 cells during bioactive agent release assay. [A] Vacuolated RPTEC cells, 24 hr culture, 400X. [B] Non-infected WI-38 cells demonstrating expected fibroblast shapes, 400X. [C] WI-38 cells 12 hrs post-exposure to spent BGM from a 24 hr RPTEC culture, 400X.
Mentions: Vacuoles were still present 24 hrs post-seeding of the RPTEC (and after the RGM change at 12 hrs) (Figure 1A), but there were no signs of contamination by extracellular bacteria or fungi. The pH at 37°C of fresh BGM was approximately 7.36 (within normal range), and ammonia was not detected using a salicylate-based method (data not shown). These findings suggested neither incorrect pH nor presence of ammonia in BGM were causing vacuolation of the RPTEC.

Bottom Line: Human coronavirus NL63 was isolated and identified by RT-PCR and sequencing, and its replication in a fresh batch of RPTEC and another type of primary human kidney cells was confirmed.Whereas we are unable to determine whether the original RPTEC were pre-infected prior to their separation from other kidney cells, or had gotten contaminated with HCoV-NL63 from an ill laboratory worker during their preparation for commercial sale, our findings are a reminder that human-derived biologicals should always be considered as potential sources of infectious agents.Importantly, HCoV-NL63 replicates to high titers in some primary human kidney cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Environmental and Global Health, College of Public Health and Health Professions, University of Florida, Box 100188, Gainesville, FL 32610-0188, USA. jlednicky@phhp.ufl.edu

ABSTRACT

Background: Cryopreserved primary human renal proximal tubule epithelial cells (RPTEC) were obtained from a commercial supplier for studies of Simian virus 40 (SV40). Within twelve hrs after cell cultures were initiated, cytoplasmic vacuoles appeared in many of the RPTEC. The RPTEC henceforth deteriorated rapidly. Since SV40 induces the formation of cytoplasmic vacuoles, this batch of RPTEC was rejected for the SV40 study. Nevertheless, we sought the likely cause(s) of the deterioration of the RPTEC as part of our technology development efforts.

Methods: Adventitious viruses in the RPTEC were isolated and/or detected and identified by isolation in various indicator cell lines, observation of cytopathology, an immunoflurorescence assay, electron microscopy, PCR, and sequencing.

Results: Cytomegalovirus (CMV) was detected in some RPTEC by cytology, an immunofluorescence assay, and PCR. Human Herpesvirus 6B was detected by PCR of DNA extracted from the RPTEC, but was not isolated. Human coronavirus NL63 was isolated and identified by RT-PCR and sequencing, and its replication in a fresh batch of RPTEC and another type of primary human kidney cells was confirmed.

Conclusions: At least 3 different adventitious viruses were present in the batch of contaminated RPTEC. Whereas we are unable to determine whether the original RPTEC were pre-infected prior to their separation from other kidney cells, or had gotten contaminated with HCoV-NL63 from an ill laboratory worker during their preparation for commercial sale, our findings are a reminder that human-derived biologicals should always be considered as potential sources of infectious agents. Importantly, HCoV-NL63 replicates to high titers in some primary human kidney cells.

Show MeSH
Related in: MedlinePlus