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      Analysis of the Aedes albopictus C6/36 genome provides insight into cell line utility for viral propagation

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          Abstract

          Background

          The 50-year-old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome.

          Results

          The C6/36 genome assembly has the largest contig N50 (3.3 Mbp) of any mosquito assembly, presents the sequences of both haplotypes for most of the diploid genome, reveals independent null mutations in both alleles of the Dicer locus, and indicates a male-specific genome. Gene annotation was computed with publicly available mosquito transcript sequences. Gene expression data from cell line RNA sequence identified enrichment of growth-related pathways and conspicuous deficiency in aquaporins and inward rectifier K + channels. As a test of utility, RNA sequence data from Zika-infected cells were mapped to the C6/36 genome and transcriptome assemblies. Host subtraction reduced the data set by 89%, enabling faster characterization of nonhost reads.

          Conclusions

          The C6/36 genome sequence and annotation should enable additional uses of the cell line to study arbovirus vector interactions and interventions aimed at restricting the spread of human disease.

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          Most cited references46

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          Isolation of a Singh's Aedes albopictus cell clone sensitive to Dengue and Chikungunya viruses.

          A Igarashi (1978)
          Twenty clones were isolated from cultured Aedes albopictus (Singh) cells in the presence of anti-Chikungunya (CHIK) virus serum. Each clone was tested for its yields of Dengue (DEN) viruses, types 1, 2, 3 and 4, and also CHIK virus. Clone C6 showed the highest yield of each virus tested. Forty-three clones obtained by recloning C6 in the presence of anti-DEN sera showed almost the same virus yields as C6. One of the clones, C6/36, showed mild to extensive cytopathic effects several days after virus infection, in contrast to the original uncloned (SAAR) cells. Fluorescent antibody staining revealed that the amount of virus antigen accumulated in the cytoplasm was almost the same in every cell in the case of clone C6/36, while it was highly heterogeneous for uncloned SAAR cells. Growth curves of the viruses indicated that clone C6/36 gave a significantly higher yield for each virus than uncloned SAAR cells up to 7 days after infection. Virus sensitivity of the C6/36 clone did not change by growing the cells with the medium used for uncloned SAAR cells, nor did the virus sensitivity of uncloned cells increase in medium used for clone C6/36. However, the C6/36 clone became resistant to CHIK virus, but not to DEN or Sindbis viruses, after incubation with the medium used for another A. albopictus cell line (SAAK). The transfer of the specific resistance to CHIK may be mediated by some latent virus related to CHIK.
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            The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line

            Summary The HeLa cell line was established in 1951 from cervical cancer cells taken from a patient, Henrietta Lacks, marking the first successful attempt to continually culture human-derived cells in vitro 1 . HeLa’s robust growth and unrestricted distribution resulted in its broad adoption – both intentionally and through widespread cross-contamination 2 – and for the past sixty years it has served a role analogous to that of a model organism 3 . Its cumulative impact is illustrated by the fact that HeLa is named in >74,000 or ~0.3% of PubMed abstracts. The genomic architecture of HeLa remains largely unexplored beyond its karyotype 4 , in part because like many cancers, its extensive aneuploidy renders such analyses challenging. We performed haplotype-resolved whole genome sequencing 5 of the HeLa CCL-2 strain, discovering point and indel variation, mapping copy-number and loss of heterozygosity (LOH), and phasing variants across full chromosome arms. We further investigated variation and copy-number profiles for HeLa S3 and eight additional strains. Surprisingly, HeLa is relatively stable with respect to point variation, accumulating few new mutations since early passaging. Haplotype resolution facilitated reconstruction of an amplified, highly rearranged region at chromosome 8q24.21 at which the HPV-18 viral genome integrated as the likely initial event underlying tumorigenesis. We combined these maps with RNA-Seq 6 and ENCODE Project 7 datasets to phase the HeLa epigenome, revealing strong, haplotype-specific activation of the proto-oncogene MYC by the integrated HPV-18 genome ~500 kilobases upstream, and permitting global analyses of the relationship between gene dosage and expression. These data provide an extensively phased, high-quality reference genome for past and future experiments relying on HeLa, and demonstrate the value of haplotype resolution for characterizing cancer genomes and epigenomes.
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              Statistics of local complexity in amino acid sequences and sequence databases

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                Author and article information

                Journal
                Gigascience
                Gigascience
                gigascience
                GigaScience
                Oxford University Press
                2047-217X
                March 2018
                10 January 2018
                10 January 2018
                : 7
                : 3
                : 1-13
                Affiliations
                [1 ]J. Craig Venter Institute, 9714 Medical Center Drive, Rockville, MD 20850, USA
                [2 ]College of Natural Sciences and Mathematics, Shepherd University, Shepherdstown, WV 25443, USA
                [3 ]Genome Informatics Section, Computational and Statistical Genomics Branch, National Human Genome Research Institute, Bethesda, MD 20892, USA
                [4 ]NCBI/NLM/NIH, 45 Center Drive, Bethesda, MD 20894, USA
                [5 ]USDA 10300 Baltimore Ave., Bldg 306 Barc-East, Beltsville, MD 20705-2350, USA
                [6 ]Department of Pathogen Biology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China
                [7 ]Department of Biochemistry and the Fralin Life Science Institute, Virginia Tech, Blacksburg, VA, USA
                [8 ]Department of Entomology and the Fralin Life Science Institute, Virginia Tech, Blacksburg, VA, USA
                [9 ]Laboratory of Ecology, Genetics and Environmental Protection, Tomsk State University, Tomsk, Russia
                [10 ]Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
                [11 ]NBACC, Fort Detrick, MD 21702, USA
                [12 ]Department of Entomology, The Ohio State University, Ohio Agricultural Research and Development Center, Wooster, OH 44691, USA
                [13 ]ATCC, 217 Perry Parkway, Gaithersburg, MD 20877, USA
                Author notes
                Corresponding address. Jason Miller, J. Craig Venter Institute, 9714 Medical Center Drive, Rockville MD 20850; E-mail: jmill02@ 123456shepherd.edu
                Article
                gix135
                10.1093/gigascience/gix135
                5869287
                29329394
                95a9c395-e13b-49ee-bfcb-968cb1d7315d
                © The Author(s) 2018. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 27 June 2017
                : 11 December 2017
                : 23 December 2017
                Page count
                Pages: 13
                Categories
                Research

                aedes albopictus,c6/36,cell line,genome assembly
                aedes albopictus, c6/36, cell line, genome assembly

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