HRT-18 Cells Research Articles

Biotinylated and radioactive cDNA probes in the detection by hybridization of bovine enteric coronavirus

cDNA, synthesized on bovine coronavirus (BCV) genomic RNA templates, could be used to detect very small quantities (i.e. 1 pg) of viral RNA by hybridization with either radioisotopic-labelled or biotinylated recombinant plasmids. Virus was optimally attached to nitrocellulose membranes when spotted in 1 × SSC, whereas 20 × SSC was superior for viral RNA. Denaturation and RNA fixation of both RNA, still encapsidated in virus particles and isolated genomic RNA, was achieved by baking of the blots in vacuum. Virus detection in the supernatant of infected HRT-18 cells was feasible, but improved significantly after proteinase K treatment. No homology was observed between virus cDNA with either plasmid DNA or nucleic acid isolated from non-infected HRT-18 cells. Hybridization with radioisotopic-labelled probes in higher formamide concentrations (up to 60%) increased the detection signals, possibly by reducing reassociation of the probe. Significant detection amplification (30–50 times) was achieved in the case of biotinylated probes by stimulation of hyperpolymer formation on already hybridized target sequences, by additional hybridization with biotinylated pUC-19. A detection amplification was also obtained when hybridization was done with two probes (pBC-52 and pBC-247), containing non-overlapping viral sequences. Although the detectability was surpassed by biotinylated probes, sensitivity was superior in radioisotopic virus detection.

Structural proteins of bovine coronavirus strain L9: effects of the host cell and trypsin treatment

SummaryThe polypeptide profile of the cell-adapted strain of bovine coronavirus (Mebus BCV-L9) is remarkably affected by the host cell and trypsin. We compared the structural proteins of virus purified from different cell lines and found cell-dependent differences in the virus structure. BCV was purified from four clones of human rectal tumour cells (HRT-18): 3 F3, D 2, 3 E 3, and 4 B 3. The structural profiles of BCV propagated in clones 3 E 3 and 3 F 3 were identical, consisting of proteins with molecular weights of 185, 160, 140, 125, 110, 100, 52, 46, 37, 31–34, and 26–28 kilodaltons (kd). BCV purified from clone D2 lacked the 100 kd species, and clone 4 B 3 yielded virus lacking the 46 kd protein. We compared the structures of BCV propagated in HRT-18 cells [BCV(HRT-18)] and virus raised in bovine fetal spleen cells [BCV(D 2 BFS)]. The concentration of the 185 kd protein was higher in BCV (D 2BFS), and it also contained a 200 kd species. Protein profiles of in vitro trypsin treated and untreated BCV(HRT-18) differed only under reducing conditions, suggesting that trypsin cleavage sites are located within disulfide-linked regions of affected proteins. Propagation of BCV in D 2 BFS cells in the presence of trypsin resulted in cleavage of the 185 kd protein and a concommitant increase of the 100 kd protein. Activation of the fusion function probably depends on this cleavage process because fusion of BCV-infected D 2 BFS cells is trypsin dependent.

Expression of bovine coronavirus antigens at the plasma membrane in infected cell cultures

Infection by bovine coronavirus (BCV) may cause severe diarrhea in neonatal calves. The virus can multiply in the human adenocarcinoma cell line HRT-18 and in bovine fetal spleen cells (BFS). Fusion of BFS cells occurs after BCV infection if trypsin is added to the culture medium. This type of fusion may be induced by other enveloped viruses following the expression of virally encoded macromolecules in the plasma membrane. The presence of viral proteins in the host plasma membrane is not, however, necessary for BCV maturation since coronaviral particles are enveloped by budding into intracellular compartments. Immunogold techniques were used to determine if BCV proteins are present in the plasma membrane of the host cell and whether their absence might explain the lack of fusion in BFS cells not treated with trypsin.Monolayers of HRT-18 cells, grown in DMEM medium with 5% fetal calf serum, were infected with BCV.

Enquête sérologique par immunofluorescence indirecte, sur les infections à coronavirus OC 43

Four-hundred seventy-two sera were examined by the indirect immunofluorescence test against Coronavirus strain OC43 adapted to the HRT18 cell culture. Sera were from children and adults hospitalized over a 4-year period, from 1979 to 1982, at the CHRU of Clermont-Ferrand (France) for different clinical reasons. Of the 446 sera tested, 61 % showed antibodies against OC43; 90 % of adult sera were positive. Coronavirus infections are very common in France. Immunity appears early in life, and infection is frequent in children from 12–36 months of age.

Plaque Assay for Titration of Bovine Enteric Coronavirus

The plaquing ability of two isolates of bovine enteric coronavirus (BECV) was studied in HRT18 (human rectal adenocarcinoma) cell monolayers. Both isolates were able to induce plaque formation within 2 to 3 days; plaques appeared as round opalescent areas which remained colourless after neutral red or crystal violet staining. A good correlation was found between the titres as determined either by counting the plaques that were visible to the naked eye before and after neutral red staining, or by enumerating fluorescence or haemadsorption foci.