This assay was performed in a similar manner to the PCR-SSCP assay described previously (section 2.6). To be able to analyse the complete HBV genome overlapping primer sets were used (see Table 2.2 for sequences and positions of primers). The primary PCR reactions were carried out as described in section 2.4.2. This was followed by a
secondary PCR which was carried out in a volume of 20 pi containing Taq polymerase
buffer, 15 mM MgCb, 70 pM dNTPs, 20 pmol of each inner primer, 2 pCi of ^^P-
deoxycytosine 5’-phosphate, 1 unit of Taq polymerase and 2 pi o f primary PCR product.
Following PCR, the products were RE digested using Hind. II or Hinc II according to
Sambrook et a l (1989) to maintain the lengths of the amplicons between 200-500 bp
CHAPTER 3
EVALUATION OF THREE TECHNIQUES USED TO INVESTIGATE V m A L HETEROGENEITY W ITHIN HBV CARRIERS.
3.1 BACKGROUND
Investigation of HBV transmission is predominantly done by HBsAg subtyping or amplification o f clinical samples by PCR, followed by direct sequencing of the amplicons. In a given investigation, if sequences of the amplicons from the putative source match
those o f the case(s) then transmission is regarded to have occurred (Lin et a l, 1990; Liang
et al., 1991; Lin et al., 1991; Hawkins et a i, 1994; Zuckerman et al., 1995; Hawkins et
al., 1996). However, direct sequencing of amplicons generated in this manner is likely to
reflect only the dominant strain present in the sample (Leitner et al., 1993; Wright et al.,
1994). Reconstitution experiments using clones from HIV-1 have shown that populations which represent less than 10-20% of the total template will not be identified if only direct
sequencing is used (Leitner et a l, 1993). This has also been shown with reconstitution
experiments using cloned material from Neisseria gonorrhoeae (Wright et a l, 1994),
which also demonstrated that direct sequencing from the same clinical isolate can produce different sequence data, and that repeated sampling yields different sequences. The data suggest that clinical samples can contain mixed sequences that potentially mask minor sequences. Direct sequencing can therefore produce reliable results on samples that have relatively homogeneous populations.
Most chronic carriers of HBV are infected with a mixture o f variants. (Okamoto et al.,
1987b; Kaneko et a l, 1989; Kew et a l, 1993). Okamoto et al. (1987b) showed in a
longitudinal study that the sequence variation in HBV genomes within an individual who contracted HBV perinatally can be greater than in someone who has carried the virus for only four years. The increase in heterogeneity within any particular carrier can be due to the fact that the virus replicates via an RNA intermediate uses a DNA polymerase with an
RT-like activity that lacks proof reading ability (Summers and Mason, 1982) (see chapter 1). Furthermore, as well as random mutations, viral mutation may also be due to immune
pressure, as has been demonstrated to occur in murine retroviruses (Pozsgay et a/., 1989).
With these considerations, and before embarking on the main body of study, it was determine if alternatives to direct sequencing would be preferable as the main tool for molecular epidemiologic investigations. One approach for studying the heterogeneity in viruses with diverse populations is limit dilution (LD), in which the dilution o f a sample
allows the amplification of single viral molecules (Simmonds et a l, 1990; Brinchmann et
al., 1991). Another approach is PCR-cloning, the isolation of a DNA fragment by
incorporation into a vector which enables it to be multiplied in bacteria (Wolinsky et a l,
1992; Scarlatti et a l, 1993). This chapter reports the investigation into how limit dilution and two PCR-cloning procedures influence the spectra of sequences recovered from HBV carriers.
3.2 SAMPLES
Details of three carriers, all surgeons, from whom sera were studied, are summarised in Table 3.1. Sera from cases of acute hepatitis (total no: 15) linked to each of the surgeons were also used in this study.
3.3 RESULTS
All the experiments were performed using primers amplifying a 256 bp fragment of the core gene between positions 1897 and 2154 (section 2.4.1). The samples were extracted
Table 3.1
Samples from surgeons who are chronic carriers of HBV.
SURGEON HBeAg STATUS ANTI-HBc IgM STATUS SURGICAL SUBSPECIALITY IMPLICATED TRANSMISSION ACTIVITY NO OF TRANSMISSION EVENTS
A positive negative CARDIOTHORACIC INTRATHORACIC
SURGERY 11
B positive negative CARDIOTHORACIC INTRATHORACIC
SURGERY . 3
C negative negative OBSTETRICS CAESAREAN
SECTION 1
Table 3.2
Quantification of circulating HBV DNA and clones from the surgeons.
SURGEON TITRE* NO OF REC
DERIVED SEQUENCES NO OF TAC DERIVED SEQUENCES NO O FLD DERIVED SEQUENCES TOTAL NUMBER OF SEQUENCES A 10" 17/20 15/20 9/50 38 B 10^ 16/20 15/20 8/50 37 C lO'* 4/20 15/20 10/50 29 in genome equivalents/ml
according to section 2.2 and submitted to PCR; the products were then cleaned (section 2.7) and submitted to cycle sequencing (section 2.8) to give the direct sequence of each sample. The HBV DNA from each of the surgeons were then subjected to T/A cloning (TAC), restriction enzyme digest cloning (REC) and limit dilution (LD).
From each sample, 20 white restriction enzyme digest cloning colonies and 20 white T/A cloning colonies were picked from the plates and screened for inserts as described in section 2.10.1 For limit dilution, PCR was carried out on 50 replicates at the end point dilution o f each sample; the limiting dilution for surgeons A, B, and C were 1:39,062,500, 1:1,562,500 and 1:312,500, respectively. Out of an original 50 replicates a maximum of 10 positive reactions were expected. The samples were further quantified by end point dilution PCR.
The number of sequences yielded per carrier are summarised in Table 3.2. These sequences were compared with the direct sequence o f the core fragment for each of the surgeons using the Jotun-Hein method of phylogenetic analysis. Both the DNA sequences and the predicted amino acid sequences were studied.
3.3.1 HBV sequence diversity in carrier A
A is regarded to be a highly infectious carrier because of his HBeAg-positive sero-status; this is also reflected by a high level o f DNA in this sample (10^ genome equivalents/ml). His virus population was relatively homogeneous; for the most part all three techniques yielded identical and near identical sequences. A single sequence, the dominant sequence, accounted for approximately 66% of the sequences isolated. This was also identical to that o f the direct sequence (figure 3.1). There were a number o f minor sequences that
Figure 3.1
Phylogeny of sequences recovered from carrier A.
DIRECT