key: cord-0296097-iukf2zmx
authors: Konishi, Kazuhiro; Yamaji, Toshiyuki; Sakuma, Chisato; Kasai, Fumio; Endo, Toshinori; Kohara, Arihiro; Hanada, Kentaro; Osada, Naoki
title: Whole-genome sequencing of Vero E6 (C1008) and comparative analysis of four Vero cell sublines
date: 2021-10-29
journal: bioRxiv
DOI: 10.1101/2021.10.26.466002
sha: cd0a6997bd4b7efae6dd1852f2185b7e0a0b1770
doc_id: 296097
cord_uid: iukf2zmx

The Vero cell line is an immortalized cell line established from kidney epithelial cells of the African green monkey. A variety of sublines have been established from the original cell line, which display different characteristics. In this study, we determined the whole-genome sequence of Vero E6 (C1008) and performed comparative analysis among Vero JCRB 0111, Vero CCL-81, Vero 76 and Vero E6. Analysis of the copy number changes and loss of heterozygosity revealed that all sublines share a large deletion and loss of heterozygosity on chromosome 12, which harbors type I interferon and CDKN2 gene clusters. We identified a substantial number of genetic differences among the sublines including single nucleotide variants, indels, and copy number variations. The spectrum of single nucleotide variants indicated a close genetic relationship between Vero JCRB0111 and Vero CCL-81, and between Vero 76 and Vero E6, and a considerable genetic gap between the former two and the latter two lines. In contrast, we confirmed the pattern of genomic integration sites of simian endogenous retroviral sequences, which was consistent among the sublines. We identified subline-specific/enriched loss of function and missense variants, which potentially contribute to the differences in response to viral infection among the Vero sublines. In particular, we focused on Vero E6-specific/enriched variants and identified four genes (IL1RAP, TRIM25, RB1CC1, and ATG2A) that contained missense variants specific or enriched in Vero E6. In addition, we found that V739I variants of ACE2, which functions as the receptor for SARS-CoV-2, were heterozygous in Vero JCRB0111, Vero CCL-81, and Vero 76; however, Vero E6 contained the allele with isoleucine, resulting from the loss of one of the X chromosomes.

Cell lines established from mammalian tissues are often used for virus isolation and culture as well 47 as for vaccine production. One of the cell lines frequently used for these purposes is the Vero cell line, insertions, and 547,598 short deletions. As expected, 94% of these variants were shared among all 127

sublines. The number of SNVs shared among sublines is presented in Figure 2 . and Vero E6 is a Vero 76-derived clone which stably has SVL27b. Furthermore, these results indicate 179 that SVL27b is a good genomic marker to distinguish the Vero 76-Vero E6 lineage from the Vero CCL-180 81 and JCBR0111 lineages (Fig. 1) . 181

We next looked into the genetic variants specifically observed or exhibiting a high frequency in 183 each subline. We defined a subline-specific/enriched variant as a variant with higher frequency 184 compared with the other sublines with statistical significance (see method were heterozygous in Vero JCRB0111, Vero CCL-81, and Vero 76, but Vero E6 harbors only the 252 isoleucine allele. The difference results from the fact that Vero E6 exhibits monosomy X. 253

In this study, we determined the whole-genome sequence of Vero E6, which has been widely used 255

for the study of SARS-CoV-2 and performed comparative genomics on four different sublines derived 256 from Vero cells. Genomic resources for the Vero cell lines will benefit quality control of vaccine-257 producing cell substrates. In addition, finding candidates genes contributing to the different 258 phenotypes of the cell lines will facilitate the identification of mechanisms of viral proliferation and 259 the development of effective and safe substrates for vaccine production. The primary goal of this study 260 was to present a whole-genome sequence of Vero E6 as research resources and catalog a list of 261 candidate variants that potentially affect the phenotypic differences among the Vero sublines. The 

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