Identification and Characterization of Epstein-Barr Virus Genomes in Lung Carcinoma Biopsy Samples by Next-Generation Sequencing Technology

Abstract

Epstein-Barr virus (EBV) has been detected in the tumor cells of several cancers, including some cases of lung carcinoma (LC). However, the genomic characteristics and diversity of EBV strains associated with LC are poorly understood. In this study, we sequenced the EBV genomes isolated from four primary LC tumor biopsy samples, designated LC1 to LC4. Comparative analysis demonstrated that LC strains were more closely related to GD1 strain. Compared to GD1 reference genome, a total of 520 variations in all, including 498 substitutions, 12 insertions, and 10 deletions were found. Latent genes were found to harbor the most numbers of nonsynonymous mutations. Phylogenetic analysis showed that all LC strains were closely related to Asian EBV strains, whereas different from African/American strains. LC2 genome was distinct from the other three LC genomes, suggesting at least two parental lineages of EBV among the LC genomes may exist. All LC strains could be classified as China 1 and V-val subtype according to the amino acid sequence of LMP1 and EBNA1, respectively. In conclusion, our results showed the genomic diversity among EBV genomes isolated from LC, which might facilitate to uncover the previously unknown variations of pathogenic significance.

Figure 1. In situ hybridization for EBER.

(A) Squamous carcinoma associated with EBV. In situ hybridization for EBER shows the brown-stained nuclei in squamous-cell nests, ×200. (B) Adenocarcinoma associated with EBV. Note the brown-stained nuclei in most of tumor cells, ×200.

Figure 2. Genetic variations among LC strains.

Circos plot demonstrates genetic variations of LC strains relative to the reference GD1 strain (AY961628). Mutations in internal repeats and terminal repeats are disregarded. The outer circle shows the positive-strand open reading frames (ORFs) (orange), repeat regions (brown), and negative-strand ORFs (blue) in the reference GD1 genome. The red, blue, and green points in the inner circles show the distributions of single nucleotide variations (SNVs), deletions, and insertions, respectively. The size of the point represents the length of the deletion or insertion.

Figure 3. Nonsynonymous mutations of LC strains.

(A) Number of nonsynonymous mutations contained in the nine categories of EBV proteins. The majority of the amino acid changes are located in latent proteins (blue) in all of the LC strains, followed by tegument proteins (red). The Latency category refers to all latent proteins expressed in EBV virus latent phase, whereas all other categories refer only to lytic proteins. (B) Amino acid changes in CD8⁺ and CD4⁺ T cell epitopes. Amino acid changes in at least one of the LC strains at known CD8⁺ and CD4⁺ T cell epitopes are marked with solid and hollow arrows, respectively.

Figure 4. Schematic diagram of variations in the 11-AA repeat and 10-AA deletion in the C-terminus of LMP1.

The pattern of the EBV-WT is shown across the top with the number indicating the amino acid (AA) positions of both sides. A black rectangle represents 11-AA (QDPDNTDDNGP) repeat element. A black rectangle with a blank window or asterisks represents 11-AA repeat element with single nucleotide variation or deletion. A blank oval represents 5-AA (HDPLP) insertion. A blank rectangle represents 10-AA (GGGHSHDSGH). Asterisks denote deletion.

Figure 5. Phylogenetic analysis of the whole EBV genomes and protein-encoding nucleotide sequences of LMP1, EBNA1, -2, -3A, BZLF1, and BLLF1 genes.

Phylogenetic analysis was performed using MEGA software (version 6.0) by Neighbour-joining (NJ) algorithm on the basis of multiple alignment of EBV strains. Bootstrap values are shown at the internal nodes.