Hepatitis Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA1
Second Department of Medicine, Nagoya City University Medical School, Nagoya 467-8601, Japan2
Author for correspondence: Betty H. Robertson. Fax +1 404 639 1563. e-mail bjr1{at}cdc.gov
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Abstract |
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It has been estimated that one-third of the projected 10 million HBV carriers in South America reside in and around the Amazon basin, including parts of Colombia, Venezuela and Peru (Fay, 1994 ). A majority of the infections in these regions are among indigenous Amerindians, with reported carrier rates of 530% (Hadler et al., 1984
; Torres & Mondolfi, 1991
; Torres, 1996
; Blitz-Dorfman et al., 1996
). The Yucpa are an indigenous group living in the Perija Mountains in western Venezuela on the border with Colombia. The Yucpa have high mortality from fulminant and rapidly progressive chronic hepatitis due to hepatitis delta virus (HDV) superinfection among the HBV carriers in this high HBV endemic population. Both HBV and HDV infection occur primarily in childhood and early adult life (Hadler et al., 1984
).
We determined complete HBV genome sequences from the sera of 12 Yucpa individuals (Table 1). The sera were obtained during serosurveys conducted during the 1980s to determine the prevalence of HBV infection in the area (Hadler et al., 1984
, 1992
). These serum samples were all positive for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in commercial radioimmunoassays (Abbott Laboratories). DNA was extracted from 50 µl serum with the MasterPure DNA purification kit (Epicentre). A full-length 3·2 kb HBV genome product was amplified by PCR by using primers P1 and P2, as described by Günther et al. (1995)
. This product was used to generate seven overlapping fragments by nested PCR amplification with primers and conditions designed for chimpanzee HBV amplification (Hu et al., 2000
) in addition to P1 and P2. Amplicons were purified by using a QIAquick purification kit (Qiagen) and sequenced by direct sequencing using dRhodamine terminators and a 377A DNA Sequencer (Applied Biosystems) by using the approach described by Hu et al. (2000)
. Sequences of one fragment (nt 561120) of VNZ8248 and VNZ8339 and another fragment (nt 18212839) of VNZ8375 could not be determined by direct sequencing of PCR products. For sequencing of these fragments, the PCR amplicons were ligated and cloned into the plasmid vector pCR II using a TA cloning kit (Invitrogen). To investigate the relationship between the sequences obtained in this study and previously reported strains, algorithms in the GCG package (Genetics Computer Group, Madison, WI, USA) were used for alignment and sequence comparison. Alignment was performed with PILEUP, followed by construction of a neighbour-joining phylogenetic tree based on genetic distances calculated using Kimuras two-parameter method (Kimura, 1980
) within GROWTREE.
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The amino acid sequences for the HBsAg protein (Fig. 2a from the Venezuelan strains were highly conserved, with only five positions of three strains containing amino acid changes (Leu9
Pro, Leu98
Val, Gln101
Arg, Leu110
Thr, Pro111
Asn). All Venezuelan sequences had Gly145, Lys122, Leu127 and Lys160, which are the amino acid determinants for subtype adw4 (Ashton-Rickardt & Murray, 1989
; Okamoto et al., 1987
; Naumann et al., 1993
; Norder et al., 1994
; Arauz-Ruiz et al., 1997
). As mentioned above, in one individual, we identified two sequences with (VNZ8248-2/VNZ8339-2) and without (VNZ8248-1/VNZ8339-1) a nine nucleotide deletion in the S region gene. This mutation resulted in a three amino acid deletion just prior to the a determinant region of HBsAg. In another individual from the same geographical region (VNZ8337), this deletion mutant was detected as a single infection, without the virus containing the complete S region genome (Table 1
).
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Antigenic epitopes in the hydrophilic central core of HBsAg, from residue 99 to 169, can be subdivided into five regions, HBs1 to HBs5 (Carman, 1997 ; Stirk et al., 1992
). Amino acid substitutions in HBs1 or HBs5 have been proposed to result in a lack of reactivity in some HBsAg assays (Carman et al., 1997
). The S region deletion that we identified in two individuals did not cause a frame shift or termination but was located in HBs1 (Fig. 2a
; highlighted in the underlined region). Deletions in the same region of HBsAg have been described in an HBsAg-negative individual whose virus was genotype D (Fig. 2a
) (Grethe et al., 1998
). However, the a determinant (HBs3 and HBs4 regions) of this virus contained numerous other mutations that could affect the immunological reactivity. The sample in which we detected an S region deletion had no detectable HBsAg serological differences from the other samples in this collection.
The amino acid sequence of the precore and core region (Fig. 2b) from the Venezuelan strains reveals that the amino acid sequences were well conserved, with a single amino acid change (Gln184
Lys) found in one sequence. One individual (VNZ8375) had two distinct core sequences, one with a deletion (VNZ8375-2) and one without a deletion (VNZ8375-1). The VNZ8375-1 sequence is identical to the core sequence in all the other Venezuelan strains within this region. The deletion of VNZ8375-2 (86 nucleotides) resulted in a frame shift and termination codon in the core region. These two strains, found in the same serum specimen, had identical sequences surrounding the deleted region, as determined by cloning. Moreover, the remaining genome sequence, determined by direct sequencing, had a single consensus sequence. This indicates that this mixed virus population did not result from a double infection and probably represents a de novo mutation within this individual.
Deletion mutants in this same region have been reported in other sequences (Fig. 2b) (Günther et al., 1996
; Preikschat et al., 1999
) and all of these deletion mutants co-exist with a sequence containing the complete core region. The majority of these deletions (which vary in length from eight to 79 amino acids) start near the major B cell epitope of hepatitis B core antigen (HBcAg) and HBeAg (HBc/e1). The HBV polymerase (P) reading frame overlaps the end of the core reading frame, and these core region deletions occur prior to the P gene start codon and do not affect the P gene reading frame. However, the deletions are predicted to interfere with the translational regulation of P protein expression by deletion of two conserved ATG codons prior to the P gene start ATG (Günther et al., 1999
). In the current model of P protein translation, the presence of these two conserved start codons, located upstream of the P gene start ATG, are responsible for suppressing P gene translation (Fouillot et al., 1993
; Hwang & Su, 1998
). Most of the core region deletion mutants that have been reported, including the Venezuelan core region deletion mutant, have deletions that include these two ATGs (data not shown). Even though the relationship between the core region deletions and regulation of P protein translation has been elucidated in vitro, the clinical significance of these mutations is still unclear. The appearance of these mutants in immunocompetent individuals is commonly associated with a favourable outcome (Marinos et al., 1996
; Nakayama et al., 1995
) while, in the absence of a humoral immune response, the outcome is less favourable (Uchida et al., 1994
; Zoulim et al., 1996
; Günther et al., 1996
), resulting in liver failure or progressive liver disease. Furthermore, the deletion mutants in patients with progressive liver disease reported by Günther et al. (1996)
were genotypes A and B, and ours is the first report of a similar deletion mutant within genotype F HBV infections.
It has long been recognized that fulminant hepatitis in Amerindian populations is a major problem (Hadler et al., 1984 ) and there is a clear link between HDV superinfection and severe disease in this population. The interaction between HDV and HBV and the effect of HDV infection on the genome of HBV is an area that has not been explored. Further studies are needed to evaluate the role of the core region deletions and their relationship to the development of fulminant hepatitis observed in these populations.
Genotype F HBV strains have been proposed to be representative of the HBV strains of Amerindian populations of the New World (Norder et al., 1994 ). The adw4 subtype, which is encoded by genotype F, has been found in French Polynesia, in Argentina, in Venezuelan Cuiva Indians (Couroucé-Pauty et al., 1983
) and in other native populations from the Amazon basin (Gaspar & Yoshida, 1987
). In this study, we have characterized 12 complete HBV sequences present in Venezuelan Yucpa Indians as genotype F and determined that the predicted subtype from the translated amino acid sequences is adw4. The sequences are most closely related to genotype F HBV derived from Colombia, a country bordering Venezuela. These 12 strains included two novel deletion mutants that have not been identified previously among genotype F strains. Our observations indicate that complete genome information, from such a limited population, may reveal unique HBV variants and provide valuable molecular information that may correlate with clinical disease features.
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Footnotes |
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b Present address: National Immunization Program, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
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References |
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Received 7 September 2000;
accepted 3 November 2000.