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.

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.

Coagulation profiling of random-source cats.

The chemistry and hemostatic parameters of class B vendor cats (Felis catus) can show wide levels of variation, possibly because of initial health status. We compared prothrombin time, partial thromboplastin time, common pathway assay and thrombin time between Class B vendor cats (n = 30) and a control group of healthy cats (n = 16). The antiprotease activities of antiXa, antiIIa, heparin cofactor II, and antithrombin were measured also. Plasma samples from citrated blood were analyzed by using standard clotting assays and commercially available chromogenic substrate assays. Tests for homogeneity of variances and 1-way ANOVA were used to test for significant differences between groups. Results of ANOVA were highly significant between groups for heparin cofactor II and Heptest activity levels. Variances were significantly different between groups for prothrombin time; therefore, an ANOVA was not done. These studies suggest that the class B cats exhibited sufficiently wide variations in their coagulation parameters that they may not be optimal subjects for vascular or cardiovascular research.