Temporal and spatial analysis of Sin Nombre virus quasispecies in naturally infected rodents

Abstract

Sin Nombre virus (SNV) is thought to establish a persistent infection in its natural reservoir, the deer mouse (Peromyscus maniculatus), despite a strong host immune response. SNV-specific neutralizing antibodies were routinely detected in deer mice which maintained virus RNA in the blood and lungs. To determine whether viral diversity played a role in SNV persistence and immune escape in deer mice, we measured the prevalence of virus quasispecies in infected rodents over time in a natural setting. Mark-recapture studies provided serial blood samples from naturally infected deer mice, which were sequentially analyzed for SNV diversity. Viral RNA was detected over a period of months in these rodents in the presence of circulating antibodies specific for SNV. Nucleotide and amino acid substitutions were observed in viral clones from all time points analyzed, including changes in the immunodominant domain of glycoprotein 1 and the 3' small segment noncoding region of the genome. Viral RNA was also detected in seven different organs of sacrificed deer mice. Analysis of organ-specific viral clones revealed major disparities in the level of viral diversity between organs, specifically between the spleen (high diversity) and the lung and liver (low diversity). These results demonstrate the ability of SNV to mutate and generate quasispecies in vivo, which may have implications for viral persistence and possible escape from the host immune system.

FIG. 1

Phylogenetic analysis of SNV G1 consensus sequences from deer mice analyzed in Table 2. Also shown are all G1 clones isolated from deer mouse Z15 C1, mark-recapture deer mice (86A943 and R2A425), and two wood rats (WR115 and WR120). The consensus sequence for each deer mouse is indicated with the animal ID number and an asterisk. SNV clones isolated from particular rodents are grouped, and sequential time points are indicated with distinct symbols and colors. NJ analysis was performed with MEGA software and the Jukes-Cantor distance estimation model. Qualitatively similar trees were generated with gamma distances (a = 0.5) for the Tamura-Nei or Kimura two-parameter distance method with either NJ or UPGMA analysis. Bootstrap analysis was carried out with 500 replicates. The percentage of bootstrap support exceeding 50% is indicated by the numbers in parenthesis next to the corresponding branches. SNV heterogeneity was demonstrated in both deer mouse and wood rat species, with similar mutation frequencies seen in all animals. Deer mouse R2A425 was seropositive for SNV for all time points analyzed. Deer mouse 86A943 was captured before (first time point) and after (all other time points) seroconversion. The deer mouse seroconverted after its first capture; however, viral sequences were amplified from all time points analyzed. The asterisk next to a viral clone from R2A425 indicates the presence of a mutation (underlined) (codon TGG→TGA) which gave rise to a stop codon at amino acid residue 81 (tryptophan), suggesting either that this clone corresponds to a defective SNV genome or that the mutation represents an error incorporated during the isolation procedure. SNV isolates CC107 and CC74 (GenBank accession no. L33474 and L33684, respectively) were also included to rule out possible contamination with viral isolates grown in the laboratory (63).

FIG. 2

Comparison of the levels of SNV diversity in different organs of a deer mouse (Z15 B7). The mutation frequencies represented in the graph are the mean mutation frequencies observed in both G1 and SVAR clones (with respect to the consensus sequence) ± standard errors, with each viral clone serving as a replicate. Statistical analysis was done on mutation frequencies with respect to organ, region, and mutation type (see Materials and Methods). A→G and U→C mutations were shown to be statistically more common than expected by chance (P < 0.0001). Non-A→G and non-U→C mutation frequencies for all organs were very similar (0.59 ± 0.22 [mean mutation frequency ± standard deviation]).

FIG. 3

Inferred amino acid alignment of G1 organ-specific SNV clones from deer mouse Z15 B7. All organ-specific viral clones are shown aligned, with the consensus sequence given at the top. Each G1 clone is listed, with the organ description on the right side of the alignment. The relative abundance of each particular sequence is given as the percentage of the total number of clones analyzed (spleen, six; liver and salivary gland, five; kidney, lung, and bladder, four; and blood, three). The immunodominant domain of G1 is shown above the consensus sequence. Amino acid mutations found in viral clones from all animals are indicated as bars above the consensus sequence for deer mouse Z15 B7 (light gray, one mutation; dark gray, two mutations; black, three mutations). The antigenic index (Jameson-Wolf; DNASTAR Inc.) for the SNV G1 region is given directly below the alignment. Possible hypervariable regions of G1 for deer mouse Z15 B7 viral clones are boxed with heavy lines, and quasineutral changes are boxed with light lines. The variability plot (VarPlot for Windows) for deer mouse Z15 B7 is shown below the antigenic index graph. Both d_N/d_S ratios and d_N − d_S values were included in the analysis. aa, amino acids.