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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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Related in: MedlinePlus

Cytopathic effects in primary HRE cells infected with HCoV-NL63/RPTEC/2004 pp A or HCoV-NL63/Amsterdam-1. [A] Non-infected confluent HRE, 400x. [B] Confluent HRE infected with HCoV-NL63/RPTEC/2004 pp A, 3 dpi, 400x. [C] Confluent RPTEC infected with HCoV-NL63/Amsterdam-1, 3 dpi, 400x. [D] Confluent LLC-MK2 cells infected with HCoV-NL63/RPTEC/2004 pp A from new RPTEC, 3 dpi, 400x.
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Figure 11: Cytopathic effects in primary HRE cells infected with HCoV-NL63/RPTEC/2004 pp A or HCoV-NL63/Amsterdam-1. [A] Non-infected confluent HRE, 400x. [B] Confluent HRE infected with HCoV-NL63/RPTEC/2004 pp A, 3 dpi, 400x. [C] Confluent RPTEC infected with HCoV-NL63/Amsterdam-1, 3 dpi, 400x. [D] Confluent LLC-MK2 cells infected with HCoV-NL63/RPTEC/2004 pp A from new RPTEC, 3 dpi, 400x.

Mentions: Newly acquired (in 2013) primary RPTEC, HRE, and HRCE cells did not release a detectable bioagent (data not shown). What may have been “owl’s eye” nuclei were observed rarely only in HRE cells. Both HCoV-NL63/RPTEC/2004 and HCoV-NL63/Amsterdam-1 caused rapid formation of CPE in RPTEC (Figure 10) and HRE cells (Figure 11) infected at a MOI of 0.1 with plaque purified HCoV-NL63/RPTEC/2004 pp A or HCoV-NL63/Amsterdam-1. We noted that the RPTEC were not vacuolated when sub-confluent (Figure 10A) yet became vacuolated once confluent (Figure 10B), but otherwise stayed viable when re-fed every 2 days with REBM. Extensive CPE consisting of rounding of the cells and cytolysis occurred by 3 dpi in RPTEC (Figure 10C-E) and 4 dpi in HRE cells (Figure 11B-C). When 1 ml of spent REBM was obtained from RPTEC or HRE cells 3 days after they had been infected with HCoV-NL63 RPTEC/2004 pp A or HCoV-NL63/Amsterdam-1, and inoculated onto LLC-MK2 cells in T25 flasks, CPE were extensive 3 days later (Figure 10F and Figure 11D). In contrast, 1 ml of spent media from non-infected (negative control) RPTEC and HRE cells had no effect on LLC-MK2 cells (data not shown). The presence of HCoV-NL63 in the spent media of RPTEC and HRE that had been inoculated with the viruses, and in the indicator LLC-MK2 that had been inoculated with spent media from the virus-infected cells, was confirmed by RT-PCR (data not shown). In contrast, CPE were sparse in HRCE cells 7 dpi with either HCoV-NL63/RPTEC/2004 pp A or HCoV-NL63/Amsterdam-1 (data not shown).


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)

Cytopathic effects in primary HRE cells infected with HCoV-NL63/RPTEC/2004 pp A or HCoV-NL63/Amsterdam-1. [A] Non-infected confluent HRE, 400x. [B] Confluent HRE infected with HCoV-NL63/RPTEC/2004 pp A, 3 dpi, 400x. [C] Confluent RPTEC infected with HCoV-NL63/Amsterdam-1, 3 dpi, 400x. [D] Confluent LLC-MK2 cells infected with HCoV-NL63/RPTEC/2004 pp A from new RPTEC, 3 dpi, 400x.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 11: Cytopathic effects in primary HRE cells infected with HCoV-NL63/RPTEC/2004 pp A or HCoV-NL63/Amsterdam-1. [A] Non-infected confluent HRE, 400x. [B] Confluent HRE infected with HCoV-NL63/RPTEC/2004 pp A, 3 dpi, 400x. [C] Confluent RPTEC infected with HCoV-NL63/Amsterdam-1, 3 dpi, 400x. [D] Confluent LLC-MK2 cells infected with HCoV-NL63/RPTEC/2004 pp A from new RPTEC, 3 dpi, 400x.
Mentions: Newly acquired (in 2013) primary RPTEC, HRE, and HRCE cells did not release a detectable bioagent (data not shown). What may have been “owl’s eye” nuclei were observed rarely only in HRE cells. Both HCoV-NL63/RPTEC/2004 and HCoV-NL63/Amsterdam-1 caused rapid formation of CPE in RPTEC (Figure 10) and HRE cells (Figure 11) infected at a MOI of 0.1 with plaque purified HCoV-NL63/RPTEC/2004 pp A or HCoV-NL63/Amsterdam-1. We noted that the RPTEC were not vacuolated when sub-confluent (Figure 10A) yet became vacuolated once confluent (Figure 10B), but otherwise stayed viable when re-fed every 2 days with REBM. Extensive CPE consisting of rounding of the cells and cytolysis occurred by 3 dpi in RPTEC (Figure 10C-E) and 4 dpi in HRE cells (Figure 11B-C). When 1 ml of spent REBM was obtained from RPTEC or HRE cells 3 days after they had been infected with HCoV-NL63 RPTEC/2004 pp A or HCoV-NL63/Amsterdam-1, and inoculated onto LLC-MK2 cells in T25 flasks, CPE were extensive 3 days later (Figure 10F and Figure 11D). In contrast, 1 ml of spent media from non-infected (negative control) RPTEC and HRE cells had no effect on LLC-MK2 cells (data not shown). The presence of HCoV-NL63 in the spent media of RPTEC and HRE that had been inoculated with the viruses, and in the indicator LLC-MK2 that had been inoculated with spent media from the virus-infected cells, was confirmed by RT-PCR (data not shown). In contrast, CPE were sparse in HRCE cells 7 dpi with either HCoV-NL63/RPTEC/2004 pp A or HCoV-NL63/Amsterdam-1 (data not shown).

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