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Role of hepatitis B virus genotype D & its mutants in occult hepatitis B infection.

Sengupta S, Panda SK, Acharya SK, Durgapal H - Indian J. Med. Res. (2013)

Bottom Line: One possible reason for this phenotype is alteration in large (L-HBsAg) to small (S-HBsAg) envelope protein ratio associated with reduced or non secretion of HBsAg.This results in quantitative levels of serum HBsAg below the detection limit of enzyme immunoassays.Pre-S2/S promoter deletions do not affect HBV replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, All India Institute of Medical Sciences, New Delhi, India.

ABSTRACT

Background & objectives: Non-detection of hepatitis B virus (HBV) envelope protein (hepatitis B surface antigen, HBsAg) in a chronically HBV infected individual has been described as occult infection. One possible reason for this phenotype is alteration in large (L-HBsAg) to small (S-HBsAg) envelope protein ratio associated with reduced or non secretion of HBsAg. This results in quantitative levels of serum HBsAg below the detection limit of enzyme immunoassays. Genotype D of HBV has a characteristic 33 nucleotide (nt) deletion upstream of the pre-S2/S promoter. This deletion may reduce HBsAg secretion in occult infection patients infected with genotype D HBV. Additional deletions in the pre-S2/S promoter may further aggravate reduced HBsAg secretion in patients infected with genotype D HBV. Thus, the aim of the present study was to determine the role of genotype D specific 33nt deletion and additional pre-S2/S promoter deletions in causing reduced or no secretion of HBsAg, in occult infection. Since these deletions overlap virus polymerase, their effect on virus replication was also investigated.

Methods: We examined the in vitro expression of HBsAg, ratio of cure and 'e' antigen (HBcAg/HBeAg), their secretion and virus replication, using overlength 1.3 mer/1.86 mer genotype A replicons, and genotype D replicons with and without additional pre-S2/S promoter deletions from cases of occult infection.

Results: Genotype D replicon showed a decrease in HBsAg secretion compared to the wild-type genotype A. Genotype D replicons carrying additional pre-S2/S promoter deletions, showed further reduction in HBsAg secretion, demonstrated presence of intracellular HBcAg/HBeAg, virus replication intermediates and 'e' antigen secretion.

Interpretation & conclusions: The characteristic 33 nt deletion of genotype D HBV reduces HBsAg secretion. Additional pre-S2/S promoter deletions may further diminish HBsAg secretion, leading to occult infection. Pre-S2/S promoter deletions do not affect HBV replication.

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(A) Ratio of extracellular to intracellular HBeAg in Huh7 cells transfected with the 313.1-1.86 and 761.1-1.86 constructs at 48 h post-transfection. (B) Intracellular expression pattern of HBcAg by indirect immunofluorescence in Huh7 cells transfected with the 313.1-1.86 and 761.1-1.86 constructs. The Figure depicts cytoplasmic staining (i, ii), nuclear staining (iii, iv) and combined cytoplasmic and nuclear staining (v, vi) of Huh7 cells transfected with the mutant 313.1-1.86 (i,iii,v) and mutant 761.1-1.86 constructs (ii, iv, vi), respectively for HBcAg.
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Figure 3: (A) Ratio of extracellular to intracellular HBeAg in Huh7 cells transfected with the 313.1-1.86 and 761.1-1.86 constructs at 48 h post-transfection. (B) Intracellular expression pattern of HBcAg by indirect immunofluorescence in Huh7 cells transfected with the 313.1-1.86 and 761.1-1.86 constructs. The Figure depicts cytoplasmic staining (i, ii), nuclear staining (iii, iv) and combined cytoplasmic and nuclear staining (v, vi) of Huh7 cells transfected with the mutant 313.1-1.86 (i,iii,v) and mutant 761.1-1.86 constructs (ii, iv, vi), respectively for HBcAg.

Mentions: Pre-core/core antigen production and processing in the overlength 313.1mer and 761.1mer replicons: HBeAg production and secretion in the overlength 313.1-1.86mer and 761.1-1.86mer replicons were observed (Fig. 3A). Cells transfected with the 313.1-1.86mer and 761.1-1.86mer (Fig. 3B) replicons showed cells with only cytoplasmic HBcAg staining (Fig. 3B-i, ii), only nuclear HBcAg staining (Fig. 3B-iii,iv) and both cytoplasmic and nuclear HBcAg staining (Fig. 3B-v, vi). These results indicate that expression of ‘e’/core antigen and secretion of ‘e’ antigen occurs in these replicons.


Role of hepatitis B virus genotype D & its mutants in occult hepatitis B infection.

Sengupta S, Panda SK, Acharya SK, Durgapal H - Indian J. Med. Res. (2013)

(A) Ratio of extracellular to intracellular HBeAg in Huh7 cells transfected with the 313.1-1.86 and 761.1-1.86 constructs at 48 h post-transfection. (B) Intracellular expression pattern of HBcAg by indirect immunofluorescence in Huh7 cells transfected with the 313.1-1.86 and 761.1-1.86 constructs. The Figure depicts cytoplasmic staining (i, ii), nuclear staining (iii, iv) and combined cytoplasmic and nuclear staining (v, vi) of Huh7 cells transfected with the mutant 313.1-1.86 (i,iii,v) and mutant 761.1-1.86 constructs (ii, iv, vi), respectively for HBcAg.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3818595&req=5

Figure 3: (A) Ratio of extracellular to intracellular HBeAg in Huh7 cells transfected with the 313.1-1.86 and 761.1-1.86 constructs at 48 h post-transfection. (B) Intracellular expression pattern of HBcAg by indirect immunofluorescence in Huh7 cells transfected with the 313.1-1.86 and 761.1-1.86 constructs. The Figure depicts cytoplasmic staining (i, ii), nuclear staining (iii, iv) and combined cytoplasmic and nuclear staining (v, vi) of Huh7 cells transfected with the mutant 313.1-1.86 (i,iii,v) and mutant 761.1-1.86 constructs (ii, iv, vi), respectively for HBcAg.
Mentions: Pre-core/core antigen production and processing in the overlength 313.1mer and 761.1mer replicons: HBeAg production and secretion in the overlength 313.1-1.86mer and 761.1-1.86mer replicons were observed (Fig. 3A). Cells transfected with the 313.1-1.86mer and 761.1-1.86mer (Fig. 3B) replicons showed cells with only cytoplasmic HBcAg staining (Fig. 3B-i, ii), only nuclear HBcAg staining (Fig. 3B-iii,iv) and both cytoplasmic and nuclear HBcAg staining (Fig. 3B-v, vi). These results indicate that expression of ‘e’/core antigen and secretion of ‘e’ antigen occurs in these replicons.

Bottom Line: One possible reason for this phenotype is alteration in large (L-HBsAg) to small (S-HBsAg) envelope protein ratio associated with reduced or non secretion of HBsAg.This results in quantitative levels of serum HBsAg below the detection limit of enzyme immunoassays.Pre-S2/S promoter deletions do not affect HBV replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, All India Institute of Medical Sciences, New Delhi, India.

ABSTRACT

Background & objectives: Non-detection of hepatitis B virus (HBV) envelope protein (hepatitis B surface antigen, HBsAg) in a chronically HBV infected individual has been described as occult infection. One possible reason for this phenotype is alteration in large (L-HBsAg) to small (S-HBsAg) envelope protein ratio associated with reduced or non secretion of HBsAg. This results in quantitative levels of serum HBsAg below the detection limit of enzyme immunoassays. Genotype D of HBV has a characteristic 33 nucleotide (nt) deletion upstream of the pre-S2/S promoter. This deletion may reduce HBsAg secretion in occult infection patients infected with genotype D HBV. Additional deletions in the pre-S2/S promoter may further aggravate reduced HBsAg secretion in patients infected with genotype D HBV. Thus, the aim of the present study was to determine the role of genotype D specific 33nt deletion and additional pre-S2/S promoter deletions in causing reduced or no secretion of HBsAg, in occult infection. Since these deletions overlap virus polymerase, their effect on virus replication was also investigated.

Methods: We examined the in vitro expression of HBsAg, ratio of cure and 'e' antigen (HBcAg/HBeAg), their secretion and virus replication, using overlength 1.3 mer/1.86 mer genotype A replicons, and genotype D replicons with and without additional pre-S2/S promoter deletions from cases of occult infection.

Results: Genotype D replicon showed a decrease in HBsAg secretion compared to the wild-type genotype A. Genotype D replicons carrying additional pre-S2/S promoter deletions, showed further reduction in HBsAg secretion, demonstrated presence of intracellular HBcAg/HBeAg, virus replication intermediates and 'e' antigen secretion.

Interpretation & conclusions: The characteristic 33 nt deletion of genotype D HBV reduces HBsAg secretion. Additional pre-S2/S promoter deletions may further diminish HBsAg secretion, leading to occult infection. Pre-S2/S promoter deletions do not affect HBV replication.

Show MeSH
Related in: MedlinePlus