Error correction and statistical analyses for intra-host comparisons of feline immunodeficiency virus diversity from high-throughput sequencing data

Abstract

BackgroundInfection with feline immunodeficiency virus (FIV) causes an immunosuppressive disease whose consequences are less severe if cats are co-infected with an attenuated FIV strain (PLV). We use virus diversity measurements, which reflect replication ability and the virus response to various conditions, to test whether diversity of virulent FIV in lymphoid tissues is altered in the presence of PLV. Our data consisted of the 3′ half of the FIV genome from three tissues of animals infected with FIV alone, or with FIV and PLV, sequenced by 454 technology.ResultsSince rare variants dominate virus populations, we had to carefully distinguish sequence variation from errors due to experimental protocols and sequencing. We considered an exponential-normal convolution model used for background correction of microarray data, and modified it to formulate an error correction approach for minor allele frequencies derived from high-throughput sequencing. Similar to accounting for over-dispersion in counts, this accounts for error-inflated variability in frequencies – and quite effectively reproduces empirically observed distributions. After obtaining error-corrected minor allele frequencies, we applied ANalysis Of VAriance (ANOVA) based on a linear mixed model and found that conserved sites and transition frequencies in FIV genes differ among tissues of dual and single infected cats. Furthermore, analysis of minor allele frequencies at individual FIV genome sites revealed 242 sites significantly affected by infection status (dual vs. single) or infection status by tissue interaction. All together, our results demonstrated a decrease in FIV diversity in bone marrow in the presence of PLV. Importantly, these effects were weakened or undetectable when error correction was performed with other approaches (thresholding of minor allele frequencies; probabilistic clustering of reads). We also queried the data for cytidine deaminase activity on the viral genome, which causes an asymmetric increase in G to A substitutions, but found no evidence for this host defense strategy.ConclusionsOur error correction approach for minor allele frequencies (more sensitive and computationally efficient than other algorithms) and our statistical treatment of variation (ANOVA) were critical for effective use of high-throughput sequencing data in understanding viral diversity. We found that co-infection with PLV shifts FIV diversity from bone marrow to lymph node and spleen.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0607-z) contains supplementary material, which is available to authorized users.

Prior Puma Lentivirus Infection Modifies Early Immune Responses and Attenuates Feline Immunodeficiency Virus Infection in Cats

“…Increased pro-inflammatory cells and mediators [ 10 ], and attenuated proviral loads in the CO vs. FIV cats [ 7 ] were seen three weeks FIV PI. However, adaptive immune responses raised against PLV were not implicated in these differences, and G to A substitutions, due to cytidine deaminase activity, were also not found to be responsible for the bottleneck of FIV infection three weeks post-PLV infection [ 11 , 12 ].…”

“…Co-infected cats also had accelerated anti-FIV capsid antibody development soon after FIV infection when compared to cats with single PLV or single FIV infection [ 9 ], and multivariate analysis implicates immediate anti-PLV immune responses involving CD8 + T cells, CD25 + cells, IL-4, IFN-γ, and the death receptor (FAS) as correlates of attenuated disease in co-infected cats [ 10 ]. Furthermore, co-infection results in a severe bottleneck restricting virulent FIV infection in the face of prior PLV infection, as evidenced by genomic analysis, suggesting a redistribution of viral infection and viral fitness [ 11 , 12 ]. Taken together, we hypothesize that the differences in available cell targets and early immune activation parameters are plausible explanations for these observations.…”