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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

RT-PCR detection of HCoV-NL63 in LLC-MK2 cells. Lane M, 100 bp MW markers (New England Biolabs); Lane 1, HCoV-NL63-specific PCR product (314 bp) amplified by PCR primers N5-PCR1 and N3-PCR1 [33]; Lane 2, HCoV-NL63-specific PCR product (237 bp) amplified by PCR primers repSZ-1 and SZ-3[33]; Lane 3, Non-infected LLC-MK2 control tested using PCR primers N5-PCR1 and N3-PCR1; Lane 4, Non-infected LLC-MK2 control tested using PCR primers repSZ-1 and SZ-3.
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Figure 5: RT-PCR detection of HCoV-NL63 in LLC-MK2 cells. Lane M, 100 bp MW markers (New England Biolabs); Lane 1, HCoV-NL63-specific PCR product (314 bp) amplified by PCR primers N5-PCR1 and N3-PCR1 [33]; Lane 2, HCoV-NL63-specific PCR product (237 bp) amplified by PCR primers repSZ-1 and SZ-3[33]; Lane 3, Non-infected LLC-MK2 control tested using PCR primers N5-PCR1 and N3-PCR1; Lane 4, Non-infected LLC-MK2 control tested using PCR primers repSZ-1 and SZ-3.

Mentions: RT-PCR and sequencing showed one of the viruses in the CV-1, HEK-293, LLC-MK2, MDCK, MDCK-London, Mv1 Lu, and Vero E6 cells was coronavirus HCoV-NL63. An example of RT-PCR reactions performed with 2 primer sets specific for HCoV-NL63 is shown in Figure 5.


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)

RT-PCR detection of HCoV-NL63 in LLC-MK2 cells. Lane M, 100 bp MW markers (New England Biolabs); Lane 1, HCoV-NL63-specific PCR product (314 bp) amplified by PCR primers N5-PCR1 and N3-PCR1 [33]; Lane 2, HCoV-NL63-specific PCR product (237 bp) amplified by PCR primers repSZ-1 and SZ-3[33]; Lane 3, Non-infected LLC-MK2 control tested using PCR primers N5-PCR1 and N3-PCR1; Lane 4, Non-infected LLC-MK2 control tested using PCR primers repSZ-1 and SZ-3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: RT-PCR detection of HCoV-NL63 in LLC-MK2 cells. Lane M, 100 bp MW markers (New England Biolabs); Lane 1, HCoV-NL63-specific PCR product (314 bp) amplified by PCR primers N5-PCR1 and N3-PCR1 [33]; Lane 2, HCoV-NL63-specific PCR product (237 bp) amplified by PCR primers repSZ-1 and SZ-3[33]; Lane 3, Non-infected LLC-MK2 control tested using PCR primers N5-PCR1 and N3-PCR1; Lane 4, Non-infected LLC-MK2 control tested using PCR primers repSZ-1 and SZ-3.
Mentions: RT-PCR and sequencing showed one of the viruses in the CV-1, HEK-293, LLC-MK2, MDCK, MDCK-London, Mv1 Lu, and Vero E6 cells was coronavirus HCoV-NL63. An example of RT-PCR reactions performed with 2 primer sets specific for HCoV-NL63 is shown in Figure 5.

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