Frequency of spontaneous mutations in an avian hepadnavirus infection

Abstract

In this study, we measured the frequency of revertants of a cytopathic strain of the duck hepatitis B virus that bears a single nucleotide substitution in the pre-S envelope protein open reading frame, resulting in the amino acid substitution G133E. Cytopathic virus mixed with known amounts of a genetically marked wild-type virus was injected into ducklings. Virus outgrowth was accompanied by a coselection of wild-type and spontaneous revertants during recovery of the ducklings from the acute liver injury caused by death of the G133E-infected cells. The frequency of individual revertants in the selected noncytopathic virus population was estimated by determining the ratio of each revertant to the wild-type virus. Spontaneous revertants were found to be present at frequencies of 1 x 10(-5) to 6 x 10(-5) per G133E genome inoculated. A mathematical model was used to estimate that the mutation rate was 0.8 x 10(-5) to 4.5 x 10(-5) per nucleotide per generation.

FIG. 1

Viremias in eight groups of ducklings. Viremias were determined by dot hybridization and phosphorimaging. The minimum viremia detectable by this method was approximately 2 × 10⁶ genomes per ml. The mean log value for all positive samples within a group for each time point was plotted.

FIG. 2

Body weights in eight groups of ducklings. All birds in each group were weighed at the indicated times, and the mean body weight at each time point was plotted.

FIG. 3

PCR assay of the emergence of WT virus in ducklings with mixed infections. Serum samples from individual infected ducks, represented in separate lanes, were amplified by PCR. The products were each mixed with 600 ng of pGEM-7Zf(+) DNA and digested with SmaI to detect the appearance of WT internal reference genomes. The upper band (C) is the control added to each reaction mixture to monitor for complete digestion. The double bands represent the undigested PCR product of G133E-derived genomes (G) and the digested product of WT internal reference genomes (W).

FIG. 4

Direct sequence of PCR products derived from representative birds in groups 2, 4, and 6. Sequencing reactions were performed on biotinylated PCR products as described in the text. The sequence ladder runs from bottom to top in the plus-strand direction. The positions of the SmaI site present in the WT internal reference and codon 133 are indicated. Arrows mark sites of potential reverting mutations. aa, amino acid.

FIG. 5

PCR assay for the genotype of virus in ducks with mixed infections. The first viremic serum sample from each group of ducklings infected with WT plus the indicated candidate revertant was amplified by PCR, and the products were mixed with pGEM DNA and digested with SmaI exactly as described for Fig. 3. The positions of the control (C), the G133E-specific (G), and the WT-specific (W) bands are indicated.

FIG. A1

Simulated growth of three virus populations in the liver of an infected duckling. (A) Numerical calculations of the fraction of liver infected with G133E (G), G133E revertant (R), and WT viruses (W). Initial conditions were m = 10⁻⁵ revertants/genome synthesized, G(0) = 10⁻³, R(0) = 10⁻⁸ [R(0) = m × G(0)], W(0) = 10⁻⁸, k_w = 1.6 day⁻¹, k_r = k_w, k_g = 3.2 day⁻¹, and k_d = 1.92 day⁻¹ [(k_g − k_d)/k_w = 0.8]. Calculations were run over a simulated time of 25 days. (B) R/W ratio during the simulation in panel A.

FIG. A2

Accumulation of revertants as a function of the growth properties of G133E. Calculations similar to those shown for Fig. A1 were performed using the indicated growth rate constant (k_g) and the net growth rate constant (k_g − k_d) for G133E. The rate constants were normalized to that of WT (k_w). The ratio of revertant to WT virus (R/W) at 25 days postinfection is plotted. Since R(0)/W(0) = 1, the ratio R/W represents the excess revertant accumulation due to growth of G through multiple generations.