1887

Abstract

We have assessed two approaches to sequencing complete human cytomegalovirus (HCMV) genomes (236 kbp) in DNA extracted from infected cell cultures (strains 3157, HAN13, HAN20 and HAN38) or clinical specimens (strains JP and 3301). The first approach involved amplifying genomes from the DNA samples as overlapping PCR products, sequencing these by the Sanger method, acquiring reads from a capillary instrument and assembling these using the Staden programs. The second approach involved generating sequence data from the DNA samples by using an Illumina Genome Analyzer (IGA), processing the filtered reads by reference-independent () assembly, utilizing the resulting sequence to direct reference-dependent assembly of the same data and finishing by limited PCR sequencing. Both approaches were successful. In particular, the investigation demonstrated the utility of IGA data for efficiently sequencing genomes from clinical samples containing as little as 3 % HCMV DNA. Analysis of the genome sequences obtained showed that each of the strains grown in cell culture was a mutant. Certain of the mutations were shared among strains from independent clinical sources, thus suggesting that they may have arisen in a common ancestor during natural infection. Moreover, one of the strains (JP) sequenced directly from a clinical specimen was mutated in two genes, one of which encodes a proposed immune-evasion function, viral interleukin-10. These observations imply that HCMV mutants exist in human infections.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.015891-0
2010-03-01
2019-09-20
Loading full text...

Full text loading...

/deliver/fulltext/jgv/91/3/605.html?itemId=/content/journal/jgv/10.1099/vir.0.015891-0&mimeType=html&fmt=ahah

References

  1. Akter, P., Cunningham, C., McSharry, B. P., Dolan, A., Addison, C., Dargan, D. J., Hassan-Walker, A. F., Emery, V. C., Griffiths, P. D. & other authors ( 2003; ). Two novel spliced genes in human cytomegalovirus. J Gen Virol 84, 1117–1122.[CrossRef]
    [Google Scholar]
  2. Arav-Boger, R., Zong, J. C. & Foster, C. B. ( 2005; ). Loss of linkage disequilibrium and accelerated protein divergence in duplicated cytomegalovirus chemokine genes. Virus Genes 31, 65–72.[CrossRef]
    [Google Scholar]
  3. Bates, M., Monze, M., Bima, H., Kapambwe, M., Kasolo, F. C., Gompels, U. A. & CIGNIS study group ( 2008; ). High human cytomegalovirus loads and diverse linked variable genotypes in both HIV-1 infected and exposed, but uninfected, children in Africa. Virology 382, 28–36.[CrossRef]
    [Google Scholar]
  4. Bradley, A. J., Kovács, I. J., Gatherer, D., Dargan, D. J., Alkharsah, K. R., Chan, P. K. S., Carman, W. F., Dedicoat, M., Emery, V. C. & other authors ( 2008; ). Genotypic analysis of two hypervariable human cytomegalovirus genes. J Med Virol 80, 1615–1623.[CrossRef]
    [Google Scholar]
  5. Bradley, A. J., Lurain, N. S., Ghazal, P., Trivedi, U., Cunningham, C., Baluchova, K., Gatherer, D., Wilkinson, G. W. G., Dargan, D. J. & Davison, A. J. ( 2009; ). High-throughput sequence analysis of variants of human cytomegalovirus strains Towne and AD169. J Gen Virol 90, 2375–2380.[CrossRef]
    [Google Scholar]
  6. Brondke, H., Schmitz, B. & Doerfler, W. ( 2007; ). Nucleotide sequence comparisons between several strains and isolates of human cytomegalovirus reveal alternate start codon usage. Arch Virol 152, 2035–2046.[CrossRef]
    [Google Scholar]
  7. Cha, T.-A., Tom, E., Kemble, G. W., Duke, G. M., Mocarski, E. S. & Spaete, R. R. ( 1996; ). Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J Virol 70, 78–83.
    [Google Scholar]
  8. Chang, W. L., Barry, P. A., Szubin, R., Wang, D. & Baumgarth, N. ( 2009; ). Human cytomegalovirus suppresses type I interferon secretion by plasmacytoid dendritic cells through its interleukin 10 homolog. Virology 390, 330–337.[CrossRef]
    [Google Scholar]
  9. Chee, M. S., Bankier, A. T., Beck, S., Bohni, R., Brown, C. M., Cerny, R., Horsnell, T., Hutchison, C. A., III, Kouzarides, T. & other authors ( 1990; ). Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154, 125–169.
    [Google Scholar]
  10. Chou, S. ( 2008; ). Cytomegalovirus UL97 mutations in the era of ganciclovir and maribavir. Rev Med Virol 18, 233–246.[CrossRef]
    [Google Scholar]
  11. Davison, A. J., Akter, P., Cunningham, C., Dolan, A., Addison, C., Dargan, D. J., Hassan-Walker, A. F., Emery, V. C., Griffiths, P. D. & Wilkinson, G. W. G. ( 2003; ). Homology between the human cytomegalovirus RL11 gene family and human adenovirus E3 genes. J Gen Virol 84, 657–663.[CrossRef]
    [Google Scholar]
  12. Dolan, A., Cunningham, C., Hector, R. D., Hassan-Walker, A. F., Lee, L., Addison, C., Dargan, D. J., McGeoch, D. J., Gatherer, D. & other authors ( 2004; ). Genetic content of wild-type human cytomegalovirus. J Gen Virol 85, 1301–1312.[CrossRef]
    [Google Scholar]
  13. Dunn, W., Chou, C., Li, H., Hai, R., Patterson, D., Stolc, V., Zhu, H. & Liu, F. ( 2003; ). Functional profiling of a human cytomegalovirus genome. Proc Natl Acad Sci U S A 100, 14223–14228.[CrossRef]
    [Google Scholar]
  14. Engelmann, I., Petzold, D. R., Kosinska, A., Hepkema, B. G., Schulz, T. F. & Heim, A. ( 2008; ). Rapid quantitative PCR assays for the simultaneous detection of herpes simplex virus, varicella zoster virus, cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6 DNA in blood and other clinical specimens. J Med Virol 80, 467–477.[CrossRef]
    [Google Scholar]
  15. Ewing, B. & Green, P. ( 1998; ). Base-calling of automated sequencer traces using Phred. II. Error probabilities. Genome Res 8, 186–194.
    [Google Scholar]
  16. Ewing, B., Hillier, L., Wendl, M. C. & Green, P. ( 1998; ). Base-calling of automated sequencer traces using Phred. I. Accuracy assessment. Genome Res 8, 175–185.[CrossRef]
    [Google Scholar]
  17. Hahn, G., Revello, M. G., Patrone, M., Percivalle, E., Campanini, G., Sarasini, A., Wagner, M., Gallina, A., Milanesi, G. & other authors ( 2004; ). Human cytomegalovirus UL131–128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes. J Virol 78, 10023–10033.[CrossRef]
    [Google Scholar]
  18. Hassan-Walker, A. F., Okwuadi, S., Lee, L., Griffiths, P. D. & Emery, V. C. ( 2004; ). Sequence variability of the α-chemokine UL146 from clinical strains of human cytomegalovirus. J Med Virol 74, 573–579.[CrossRef]
    [Google Scholar]
  19. Huber, M. T., Tomazin, R., Wisner, T., Boname, J. & Johnson, D. C. ( 2002; ). Human cytomegalovirus US7, US8, US9, and US10 are cytoplasmic glycoproteins, not found at cell surfaces, and US9 does not mediate cell-to-cell spread. J Virol 76, 5748–5758.[CrossRef]
    [Google Scholar]
  20. Katoh, K., Kuma, K., Toh, H. & Miyata, T. ( 2005; ). mafft version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res 33, 511–518.[CrossRef]
    [Google Scholar]
  21. Kotenko, S. V., Saccani, S., Izotova, L. S., Mirochnitchenko, O. V. & Pestka, S. ( 2000; ). Human cytomegalovirus harbors its own unique IL-10 homolog (cmvIL-10). Proc Natl Acad Sci U S A 97, 1695–1700.[CrossRef]
    [Google Scholar]
  22. Li, H., Ruan, J. & Durbin, R. ( 2008; ). Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res 18, 1851–1858.[CrossRef]
    [Google Scholar]
  23. Lockridge, K. M., Zhou, S. S., Kravitz, R. H., Johnson, J. L., Sawai, E. T., Blewett, E. L. & Barry, P. A. ( 2000; ). Primate cytomegaloviruses encode and express an IL-10-like protein. Virology 268, 272–280.[CrossRef]
    [Google Scholar]
  24. Lurain, N. S., Fox, A. M., Lichy, H. M., Bhorade, S. M., Ware, C. F., Huang, D. D., Kwan, S.-P., Garrity, E. R. & Chou, S. ( 2006; ). Analysis of the human cytomegalovirus genomic region from UL146 through UL147A reveals sequence hypervariability, genotypic stability, and overlapping transcripts. Virol J 3, 4 [CrossRef]
    [Google Scholar]
  25. Mandic, L., Miller, M. S., Coulter, C., Munshaw, B. & Hertel, L. ( 2009; ). Human cytomegalovirus US9 protein contains an N-terminal signal sequence and a C-terminal mitochondrial localization domain, and does not alter cellular sensitivity to apoptosis. J Gen Virol 90, 1172–1182.[CrossRef]
    [Google Scholar]
  26. Mattick, C., Dewin, D., Polley, S., Sevilla-Reyes, E., Pignatelli, S., Rawlinson, W., Wilkinson, G., Dal Monte, P. & Gompels, U. A. ( 2004; ). Linkage of human cytomegalovirus glycoprotein gO variant groups identified from worldwide clinical isolates with gN genotypes, implications for disease associations and evidence for N-terminal sites of positive selection. Virology 318, 582–597.[CrossRef]
    [Google Scholar]
  27. McSharry, B. P., Tomasec, P., Neale, M. L. & Wilkinson, G. W. G. ( 2003; ). The most abundantly transcribed human cytomegalovirus gene (β2.7) is non-essential for growth in vitro. J Gen Virol 84, 2511–2516.[CrossRef]
    [Google Scholar]
  28. Murphy, E., Yu, D., Grimwood, J., Schmutz, J., Dickson, M., Jarvis, M. A., Hahn, G., Nelson, J. A., Myers, R. M. & Shenk, T. E. ( 2003; ). Coding potential of laboratory and clinical strains of human cytomegalovirus. Proc Natl Acad Sci U S A 100, 14976–14981.[CrossRef]
    [Google Scholar]
  29. Pignatelli, S., Dal Monte, P., Rossini, G. & Landini, M. P. ( 2004; ). Genetic polymorphisms among human cytomegalovirus (HCMV) wild-type strains. Rev Med Virol 14, 383–410.[CrossRef]
    [Google Scholar]
  30. Prichard, M. N., Penfold, M. E. T., Duke, G. M., Spaete, R. R. & Kemble, G. W. ( 2001; ). A review of genetic differences between limited and extensively passaged human cytomegalovirus strains. Rev Med Virol 11, 191–200.[CrossRef]
    [Google Scholar]
  31. Puchhammer-Stöckl, E. & Görzer, I. ( 2006; ). Cytomegalovirus and Epstein-Barr virus subtypes – the search for clinical significance. J Clin Virol 36, 239–248.[CrossRef]
    [Google Scholar]
  32. Rasmussen, L., Geissler, A. & Winters, M. ( 2003; ). Inter- and intragenic variations complicate the molecular epidemiology of human cytomegalovirus. J Infect Dis 187, 809–819.[CrossRef]
    [Google Scholar]
  33. Ryckman, B. J., Chase, M. C. & Johnson, D. C. ( 2008; ). HCMV gH/gL/UL128–131 interferes with virus entry into epithelial cells: evidence for cell type-specific receptors. Proc Natl Acad Sci U S A 105, 14118–14123.[CrossRef]
    [Google Scholar]
  34. Sekulin, K., Görzer, I., Heiss-Czedik, D. & Puchhammer-Stöckl, E. ( 2007; ). Analysis of the variability of CMV strains in the RL11D domain of the RL11 multigene family. Virus Genes 35, 577–583.[CrossRef]
    [Google Scholar]
  35. Sinzger, C., Hahn, G., Digel, M., Katona, R., Sampaio, K. L., Messerle, M., Hengel, H., Koszinowski, U., Brune, W. & Adler, B. ( 2008; ). Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E. J Gen Virol 89, 359–368.[CrossRef]
    [Google Scholar]
  36. Staden, R., Beal, K. F. & Bonfield, J. K. ( 2000; ). The Staden package, 1998. Methods Mol Biol 132, 115–130.
    [Google Scholar]
  37. Stanton, R., Westmoreland, D., Fox, J. D., Davison, A. J. & Wilkinson, G. W. G. ( 2005; ). Stability of human cytomegalovirus genotypes in persistently infected renal transplant recipients. J Med Virol 75, 42–46.[CrossRef]
    [Google Scholar]
  38. Tomasec, P., Braud, V. M., Rickards, C., Powell, M. B., McSharry, B. P., Gadola, S., Cerundolo, V., Borysiewicz, L. K., McMichael, A. J. & Wilkinson, G. W. G. ( 2000; ). Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science 287, 1031–1033.[CrossRef]
    [Google Scholar]
  39. Wandinger, K.-P., Jabs, W., Siekhaus, A., Bubel, S., Trillenberg, P., Wagner, H.-J., Wessel, K., Kirchner, H. & Hennig, H. ( 2000; ). Association between clinical disease activity and Epstein-Barr virus reactivation in MS. Neurology 55, 178–184.[CrossRef]
    [Google Scholar]
  40. Yan, H., Koyano, S., Inami, Y., Yamamoto, Y., Suzutani, T., Mizuguchi, M., Ushijima, H., Kurane, I. & Inoue, N. ( 2008; ). Genetic linkage among human cytomegalovirus glycoprotein N (gN) and gO genes, with evidence for recombination from congenitally and post-natally infected Japanese infants. J Gen Virol 89, 2275–2279.[CrossRef]
    [Google Scholar]
  41. Zerbino, D. R. & Birney, E. ( 2008; ). Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18, 821–829.[CrossRef]
    [Google Scholar]
  42. Zhou, F., Li, Q. & Gao, S. J. ( 2009; ). A sequence-independent in vitro transposon-based strategy for efficient cloning of genomes of large DNA viruses as bacterial artificial chromosomes. Nucleic Acids Res 37, e2 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.015891-0
Loading
/content/journal/jgv/10.1099/vir.0.015891-0
Loading

Data & Media loading...

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error