key: cord-0300044-gcgpa3ir authors: Quinteros, José A.; Browning, Glenn F.; Noormohammadi, Amir H.; Stevenson, Mark A.; Coppo, Mauricio J. C.; Loncoman, Carlos A.; Ficorilli, Nino; Lee, Sang-Won; Diaz-Méndez, Andrés title: Serum-free medium increases the replication rate of the avian coronavirus infectious bronchitis virus in chicken embryo kidney cells date: 2021-04-30 journal: bioRxiv DOI: 10.1101/2021.04.30.442100 sha: e19318d7154610df355c2918197b89aefec93af9 doc_id: 300044 cord_uid: gcgpa3ir Infectious bronchitis virus (IBV), an avian coronavirus, can be isolated and cultured in tracheal organ cultures (TOCs), embryonated eggs and cell cultures. TOCs and embryonated eggs are commonly used for viral isolation but use of these is laborious and expensive. Cell cultures have been used only with IBV strains that have previously been adapted to grow under laboratory conditions, and not for primary isolation. Previous studies using the coronavirus porcine epidemic diarrhoea virus (PEDV) have suggested that foetal bovine serum (FBS), a common component of cell culture media, can inhibit the adsorption of coronaviruses onto the host cell membrane receptors. In the present study, the replication of IBV in primary chicken embryo kidney (CEK) cell cultures and the Leghorn hepatocellular carcinoma (LMH) cell line was examined using two different cell culture media, one containing FBS and the other containing yeast extract (YE). A reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay was used to quantify viral RNA copies in cell lysates. The highest concentrations of viral genomes were observed when the cell culture medium did not contain FBS. Examination of the infectivity of virus grown in CEK cell cultures was examined by titration in embryonated chicken eggs, demonstrating that the cell lysate from CEK cell cultures in medium without FBS contained a higher median embryo infectious dose (EID50) than that from CEK cell cultures in medium containing FBS. These results suggest that improved replication of IBV in cell cultures can be achieved by the omission of FBS from the cell culture medium. This may enhance the potential for production of vaccines in cell culture and facilitate the isolation of emergent IBV strains in cell cultures. The cDNA was then assayed using two different qPCRs. The first qPCR targeted the gene of interest 161 (GOI), the N protein-3′UTR region of the IBV genome, and the second qPCR targeted a housekeeping 162 gene (HKG), the mRNA of the chicken GAPDH gene (Table 1) Differences were considered significant when P < 0.05. In the experiment using LMH cells, the effect was similar. There were no differences between 1, 24 220 and 72 HPI in any of the treatments containing FBS. The biggest increase in viral concentration was 221 seen in those cultures that were infected using the cell lysate from a CEK cell culture with the 214 Differences were considered significant when P < 0.05. Titres of IBV VicS-v cultured in CEK cells 230 The greatest differences in viral RNA concentrations between 1 and 24 HPI were detected when the 231 NT inoculum and NT medium (NTINTM) were used, and when the medium was supplemented with YE 232 rather than FBS (Table 2) . Therefore, lysates from NTINTM cultures were used to compare titres of 233 viable virus obtained using media containing FBS or YE. As shown in Table 4 , the titres at 1 HPI were 234 similar in the cultures with FBS (1.08 × 10 1 EID 50 per ml) or without FBS (1.47 × 10 1 EID 50 per ml). However, at 24 HPI the viral titres in the cultures with FBS had increased to 0.68 × 10 3 EID 50 per ml, 236 while in the cultures with YE (without FBS) the titres had increased to 5.01 × 10 3 EID 50 per ml (Table 237 4). The geometric mean titres were calculated using the assessments of the three independent 238 observers. The titres were compared using a one-way ANOVA and Tukey's multiple comparison test. 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