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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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Related in: MedlinePlus

(A) Huh7 cells transfected with the 313.1-1.86 and 761.1-1.86 constructs depicts Southern hybridization of intracellular DNA (Fig. 4A) at 72 h post-transfection. NS: Non specific DNA, Mo:Mock transfection, RDN: cells transfected with replication deficient negative control plasmid pRLΔ(77-1246)WTSPGE, M: 100pg marker consisting of linear double stranded fragments of HBV DNA, not shown, 313.1 : cells transfected with 313.1-1.86 construct, 761.1: cells transfected with 761.1-1.86 construct, RC: relaxed circular DNA. DL: double stranded linear DNA. The smear indicates HBV replication intermediates. (B) Relative levels of intracellular HBV DNA in Huh7 cells transfected with 313.1-1.86 and 761.1-1.86 constructs, by Taqman based quantitative real time PCR at 72 h post-transfection. The HBV DNA copy number fold over background normalized for transfection efficiency is depicted.
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Figure 4: (A) Huh7 cells transfected with the 313.1-1.86 and 761.1-1.86 constructs depicts Southern hybridization of intracellular DNA (Fig. 4A) at 72 h post-transfection. NS: Non specific DNA, Mo:Mock transfection, RDN: cells transfected with replication deficient negative control plasmid pRLΔ(77-1246)WTSPGE, M: 100pg marker consisting of linear double stranded fragments of HBV DNA, not shown, 313.1 : cells transfected with 313.1-1.86 construct, 761.1: cells transfected with 761.1-1.86 construct, RC: relaxed circular DNA. DL: double stranded linear DNA. The smear indicates HBV replication intermediates. (B) Relative levels of intracellular HBV DNA in Huh7 cells transfected with 313.1-1.86 and 761.1-1.86 constructs, by Taqman based quantitative real time PCR at 72 h post-transfection. The HBV DNA copy number fold over background normalized for transfection efficiency is depicted.

Mentions: Replication of 313.1-1.86mer and 761.1-1.86mer replicons: The mutant 313.1-1.86mer and 761.1-1.86mer replicons showed an approximately 3.2 kb intracellular double stranded linear virus DNA with a smear of virus replication intermediates by Southern blot. Relaxed circular virus DNA migrating slightly slower than linear double stranded DNA was also observed in case of the 761.1-1.86mer replicon (Fig. 4A). Replicons 313.1-1.86 and 761.1-1.86 showed approximately thousand fold greater DNA compared to cells transfected with pRLΔ(77-1246)WTSPGE (negative control-HBV DNA coding for HBV Large, Middle, Small-HBsAg promoters, genes and enhancers I,II)9 in the corresponding real time PCR experiment (Fig. 4B). Thus, deletions in the spacer region of virus polymerase of mutant 313.1-1.86 mer and 761.1-1.86mer replicons do not affect virus replication.


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) Huh7 cells transfected with the 313.1-1.86 and 761.1-1.86 constructs depicts Southern hybridization of intracellular DNA (Fig. 4A) at 72 h post-transfection. NS: Non specific DNA, Mo:Mock transfection, RDN: cells transfected with replication deficient negative control plasmid pRLΔ(77-1246)WTSPGE, M: 100pg marker consisting of linear double stranded fragments of HBV DNA, not shown, 313.1 : cells transfected with 313.1-1.86 construct, 761.1: cells transfected with 761.1-1.86 construct, RC: relaxed circular DNA. DL: double stranded linear DNA. The smear indicates HBV replication intermediates. (B) Relative levels of intracellular HBV DNA in Huh7 cells transfected with 313.1-1.86 and 761.1-1.86 constructs, by Taqman based quantitative real time PCR at 72 h post-transfection. The HBV DNA copy number fold over background normalized for transfection efficiency is depicted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: (A) Huh7 cells transfected with the 313.1-1.86 and 761.1-1.86 constructs depicts Southern hybridization of intracellular DNA (Fig. 4A) at 72 h post-transfection. NS: Non specific DNA, Mo:Mock transfection, RDN: cells transfected with replication deficient negative control plasmid pRLΔ(77-1246)WTSPGE, M: 100pg marker consisting of linear double stranded fragments of HBV DNA, not shown, 313.1 : cells transfected with 313.1-1.86 construct, 761.1: cells transfected with 761.1-1.86 construct, RC: relaxed circular DNA. DL: double stranded linear DNA. The smear indicates HBV replication intermediates. (B) Relative levels of intracellular HBV DNA in Huh7 cells transfected with 313.1-1.86 and 761.1-1.86 constructs, by Taqman based quantitative real time PCR at 72 h post-transfection. The HBV DNA copy number fold over background normalized for transfection efficiency is depicted.
Mentions: Replication of 313.1-1.86mer and 761.1-1.86mer replicons: The mutant 313.1-1.86mer and 761.1-1.86mer replicons showed an approximately 3.2 kb intracellular double stranded linear virus DNA with a smear of virus replication intermediates by Southern blot. Relaxed circular virus DNA migrating slightly slower than linear double stranded DNA was also observed in case of the 761.1-1.86mer replicon (Fig. 4A). Replicons 313.1-1.86 and 761.1-1.86 showed approximately thousand fold greater DNA compared to cells transfected with pRLΔ(77-1246)WTSPGE (negative control-HBV DNA coding for HBV Large, Middle, Small-HBsAg promoters, genes and enhancers I,II)9 in the corresponding real time PCR experiment (Fig. 4B). Thus, deletions in the spacer region of virus polymerase of mutant 313.1-1.86 mer and 761.1-1.86mer replicons do not affect virus replication.

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