Genetic Linkage of Hepatitis B Virus in Peripheral Blood Leukocytes Provides Evidence for Contamination

Abstract

Recently, Datta et al. proposed that hepatitis B virus (HBV) invariably infects peripheral blood lymphocytes (PBLs) and that the HBV sequences obtained from PBLs differ from those recovered from the serum due to major differences in humoral immune pressure (2). The basis for their conclusion was a high prevalence of a mutation at G145R in the HBV of the PBL compartment, which was not found in sera. We believe that these data result from contamination of the PBL sequences. Learn et al. proposed guidelines for protecting the integrity of the HIV sequence databases (4). They suggested examining a distance matrix for “high levels of unexpected similarity” that would raise suspicion for contamination. A BLAST search including sequences from the Datta et al. paper confirmed identity of a number of the PBL sequences but also identified another identical sequence (GenBank accession number {type:entrez-nucleotide,attrs:{text:DQ084757,term_id:68138239,term_text:DQ084757}}DQ084757). Interestingly, that sequence was published in 2006 from the same investigators (1) and was designated X2. The X2 sequence is identical to many of their PBL sequences in the current report, including PB06, PB16, PB24, PB30, PB35, PB38, PB39, PB43, PB45, PB47, and PB54 (Fig. ​(Fig.1).1). Only mixed bases separate X2 from other PBL sequences, including PB02, PB33, PB41, PB61, PB62, and PB76, indicating that these also most likely contain X2. A single nucleotide change separates X2 from PB17 and PB26, as well as a second cluster involving Y3 and PB07, which, in turn, are only one mixed base different from PB19 and PB32. Furthermore, the G145R mutation, which they attribute to shared immune escape in the PBL compartment, was also found in several sequences from the 2006 paper, including X2 and Y3 (which were, according to Table 1 in the 2006 publication, obtained from siblings). The variability seen in Fig. ​Fig.11 of that publication also refutes the hypothesis that constraints on HBV evolution at the studied locus explain the homogeneity of their sequences. FIG. 1. Network analysis of highly similar HBV sequences from two publications. Twenty-two HBV sequences (PBxx) reportedly amplified from peripheral blood leukocyte DNA described in a recent publication (2), depicted with two sequences (X2 and Y3) described in ... Their data seem biologically implausible since they indicate that the HBV in the serum and that in the PBL had different genotypes. For this to occur, one would have to hypothesize that the subjects were infected with two different genotypes but that only one of the genotypes was able to enter PBLs and subsequently disappeared from the circulating quasispecies. We raise these concerns because we strongly believe that inferences about HBV biology, and the integrity of the HBV database, need to be protected from sequences generated through contamination. Though the authors state that “the possible cross-contamination between samples or reagents was ruled out by including appropriate controls during all of the experimental steps and aptly following the PCR recommendations of Kwok and Higuchi” (3), simply applying these precautions does not always prevent contamination. The HBV community needs to remain vigilant for sequences generated by contamination; this goal would be served by following the principles proposed by Learn et al. (4). Unexpected homogeneity is strongly indicative of tight epidemiological linkage or contamination. In the case of the paper by Datta et al., only the latter seems plausible.