Elsevier

Virus Research

Volume 156, Issues 1–2, March 2011, Pages 113-120
Virus Research

Full genome sequencing and analysis of human cytomegalovirus strain JHC isolated from a Korean patient

https://doi.org/10.1016/j.virusres.2011.01.005Get rights and content

Abstract

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen and contains double stranded DNA genome with approximately 230 kbp. Because of its huge size, comparative genomic studies of HCMV genome have been limited. In this study it was attempted to obtain and analyze the full genome sequence from clinical isolate from Korea. The strain JHC was isolated from Korean patient undergoing bone marrow transplantation who exhibited resistance to ganciclovir treatment (Lee et al., 2005). The virus was plaque-purified, and the full genome sequence was determined by pyrosequencing technique. The JHC genome was found to contain 235,476 bp and 165 open reading frames (ORFs). Comparison with the full genome nucleotide sequences of 11 other HCMV strains suggest that JHC is not closely related with any other strains at genome level. As expected, JHC lacked IRL sequences found in lab-adapted AD169-varUK strain and this region was replaced by ORFs UL133–UL150 as in other clinical isolates. Two ORFs (UL1 and UL119) of the strain JHC were found to be truncated due to early stop codons, and RL6 contains an unusual start codon TTG. The strain JHC contains all the genetic information for micro RNAs known to be present in HCMV.

Introduction

Human cytomegalovirus (HCMV) belongs to beta-herpesvirus and contains double-stranded DNA with 230–240 kbp, largest of the human viruses. HCMV is a ubiquitous pathogen and found worldwide both in developed industrial societies and in isolated aboriginal groups (Landolfo et al., 2003). Although most of the HCMV infections are asymptomatic, severe clinical outcomes may arise in neonates, immunocompromised hosts and recipients of organ transplantation (Landolfo et al., 2003, Mocarski et al., 2007).

Since its first successful isolation from the salivary glands, adenoid tissues and urine in 1950s (Rowe et al., 1956, Smith, 1956, Weller et al., 1957), HCMV have been isolated worldwide from various parts of the infected human. Genome-wide analyses of the HCMV genetic contents became possible when the genome of the widely used laboratory strain AD169 was fully sequenced (Chee et al., 1990) by plasmid cloning and Sanger method. Analysis of its 230 kbp genome has identified ∼150 to at most up to 192 open reading frames (ORFs) with the potential to encode a protein (Chee et al., 1990, Davison et al., 2003b, Murphy et al., 2003a). Most low-passaged strains contain 19 ORFs encompassing ∼15 kbp in UL/b′ region that are absent from the highly passaged strain AD169 (Cha et al., 1996). This region is inverted within the genome relative to its counterpart in AD169.

Obtaining the whole genome sequences of HCMV has been hampered due to the large genome size of HCMV. Lately sequencing technologies have developed and almost 20 whole genome sequences have been determined for HCMV. Cloning of the whole HCMV genome into bacterial artificial chromosome (Borst et al., 1999) enabled full genome sequences of well-known strains such as Toledo (Murphy et al., 2003b), Towne (Dunn et al., 2003, Murphy et al., 2003b), TB40/E (Sinzger et al., 2008), and clinical isolates FIX, PH and TR (Murphy et al., 2003b). Recently high-throughput sequencing technology using Illumina Genome Analyzer has been used to obtain complete genome sequences of Towne and AD169 variants as well as several clinical isolates (Bradley et al., 2009, Cunningham et al., 2010). PCR sequencing technology has been applied to determine the whole genome sequences for clinical isolates (Cunningham et al., 2010, Dargan et al., 2010).

Until now, all of the full genome sequences of HCMV isolates are from European countries or the United States of America and full genome sequences of HCMV isolated from Asian people have not been reported. In this study, we report isolation of a clinical strain of HCMV from a Korean patient and its full genome sequencing and partial characterization of the ORFs.

Section snippets

Virus and DNA sequencing

The HCMV strain JHC was isolated in 2003 from a Korean patient undergoing bone marrow transplantation. The patient exhibited resistance to ganciclovir treatment, but the isolated virus was sensitive to ganciclovir and did not contain M460V mutation in UL97 gene (Lee et al., 2005). The virus from the patient's blood was inoculated into human foreskin fibroblast (HFF) cells and the plaque was purified and underwent three additional passages in HFF cells. The virus used for sequencing in this

JHC genome architecture and ORF mapping

The genome of the strain JHC was identified to be 235,476 bp long. The architecture of the JHC genome is typical of HCMV in that the genome could be divided into six regions, TRL, UL, IRL, IRS, US and TRS and the length of each region is 940 bp, 193,457 bp, 940 bp, 2639 bp, 35,512 bp and 2639 bp long, respectively. Genome map of the strain JHC is presented in Fig. 1A. Overall genome length and architecture are very similar among HCMV strains except that the strain AD169-varUK contains much larger TRL

Discussion

In this manuscript, we report isolation and full genome sequencing of the strain JHC from Korean patient. The virus was isolated from a bone-marrow transplantation patient who exhibited resistance to ganciclovir treatment. However, the isolated virus was found to be sensitive to ganciclovir (Lee et al., 2005), may be due to selection of minor viral species with a growth advantage in cell culture. The virus was named JHC and the full genome sequence was determined this time. The strain JHC is

Acknowledgement

This work was supported by the Korea Research Foundation Grant funded by the Korean Government (Ministry of Education, Science and Technology) (The Regional Research Universities Program/Chungbuk BIT Research-Oriented University Consortium).

References (27)

  • F. Grey et al.

    Identification and function of human cytomegalovirus microRNAs

    J. Clin. Virol.

    (2008)
  • G. Hahn et al.

    Cloning of the genomes of human cytomegalovirus strains Toledo TownevarRIT3, and Towne long as BACs and site-directed mutagenesis using a PCR-based technique

    Virology

    (2003)
  • S. Landolfo et al.

    The human cytomegalovirus

    Pharmacol. Ther.

    (2003)
  • P. Akter et al.

    Two novel spliced genes in human cytomegalovirus

    J. Gen. Virol.

    (2003)
  • E.M. Borst et al.

    Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants

    J. Virol.

    (1999)
  • A.J. Bradley et al.

    High-throughput sequence analysis of variants of human cytomegalovirus strains Towne and AD169

    J. Gen. Virol.

    (2009)
  • T.A. Cha et al.

    Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains

    Virology

    (1996)
  • M.S. Chee et al.

    Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169

    Curr. Top. Microbiol. Immunol.

    (1990)
  • C. Cunningham et al.

    Sequences of complete human cytomegalovirus genomes from infected cell cultures and clinical specimens

    J. Gen. Virol.

    (2010)
  • D.J. Dargan et al.

    Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture

    J. Gen. Virol.

    (2010)
  • A.J. Davison et al.

    Homology between the human cytomegalovirus RL11 gene family and human adenovirus E3 genes

    J. Gen. Virol.

    (2003)
  • A.J. Davison et al.

    The human cytomegalovirus genome revisited: comparison with the chimpanzee cytomegalovirus genome

    J. Gen. Virol.

    (2003)
  • L. Dölken et al.

    Cytomegalovirus microRNAs

    Virus Genes

    (2005)
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