Intersegment Recombination During Influenza A Virus Replication Gives Rise to a Novel Class of Defective Viral Genomes

Abstract

Influenza A virus (IAV) is a highly diverse pathogen with genetic variability primarily driven by mutation and reassortment. Using next-generation sequencing (NGS), we characterised defective viral genomes (DVGs) generated during the serial passaging of influenza A/Puerto Rico/8/1934 (H1N1) virus in embryonated chicken eggs. Deletions were the most abundant DVG type, predominantly accumulating in the polymerase-encoding segments. Notably, we identified and validated a novel class of multisegment DVGs arising from intersegment recombination events, providing evidence that the IAV RNA polymerase can detach from one genomic template and resume synthesis on another. Multisegment recombination primarily involved segments 1-3 but also occurred between other segment pairings. In specific lineages, certain multisegment DVGs reached high frequencies and persisted through multiple passages, suggesting they are not transient by-products of recombination but may possess features that support stable maintenance. Furthermore, multisegment DVGs were shown to be encapsidated within virions, similar to deletion DVGs. The observation of recombination between segments with limited sequence homology underscores the potential for complex recombination to expand IAV genetic diversity. These findings suggest recombination-driven DVGs represent a previously underappreciated mechanism in influenza virus evolution.

Keywords: RNA recombination; defective viral genomes; influenza A virus; multisegment recombination; next-generation sequencing; viral evolution.

Figure 1

DVG recombination events identified by ViReMa analysis of Illumina sequencing data from Influenza A/PR8 infections. Embryonated chicken eggs were inoculated with a stock of influenza A/PR8 virus that had been diluted by a factor of 10⁻⁶. Bars represent the mean ± standard deviation (SD) of normalised junction read counts (reads per million ×) across three biological replicates (N = 3), each with three technical replicates derived from independent sequencing runs of the same sample. Statistical comparison between deletion and multisegment recombination events was performed using a paired t-test (df = 2.000, p = 0.012 *).

Figure 2

Heatmaps representing the frequency of recombination events across the eight segments of influenza A/PR8: (A) deletion DVGs and (B) multisegment DVGs. Embryonated chicken eggs were inoculated with a stock of influenza A/PR8 virus diluted 10⁻⁶. The data represent the mean of three biological replicates (N = 3), each with three technical replicates derived from independent sequencing runs of the same sample. Colour intensity corresponds to the sum of normalised junction read counts (reads per million ×). For deletion DVGs, one-way ANOVA followed by Tukey’s HSD post hoc test was performed to assess statistical differences in normalised read counts across segments. Full statistical details for deletion events are provided in Table A2. For multisegment DVGs, Welch’s t-test was conducted to determine significant differences in recombination frequency between polymerase-encoding segments (1–3) and segments (4–8).

Figure 3

Schematic diagram illustrating the assembly of a multisegment DVG formed through recombination between segments 1 and 2 of Influenza A Virus. A short region of sequence overlap (microhomology) facilitates the junction between the two distinct segments. In this example, the junction occurs between nucleotides 279–283 of segment 1 and 2203–2207 of segment 2, resulting in a multisegment DVG with a total length of 417 nucleotides, as previously outlined in Table 1. Conserved terminal regions from segments 1 and 2 are retained, with the overlapping sequence highlighted.

Figure 4

Persistence of multisegment DVGs during serial passaging of Influenza A Virus in embryonated chicken eggs. An IAV stock (Stock 6) was serially passaged seven times in embryonated chicken eggs, with the virus diluted 10⁻⁶ from the previous passage at each step. (A) The number of normalised reads that contained a deletion junction and the number of normalised reads containing a multisegment junction at each of the passage of the IAV stock. (B) The number of unique multisegment DVGs from the original Stock 6 (passage 0) that persisted at each subsequent passage. (C) The proportion of multisegment DVGs at each passage that were also detected in the preceding passage. (D) The abundances of three unique multisegment DVGs (represented by different colours) were selected based on being the most abundant multisegment species in at least one passage amongst the retained mulitsegment DVGs. Two of these DVGs, [S4 +1578]–[S3 −464] (green) and [S1 +1330]–[S4 −1310] (purple), showed significant correlations with the overall abundance of multisegment DVGs shown in (A) (Pearson’s R² = 0.6255 and 0.5864; p = 0.0194 and 0.0268, respectively).

Figure 5

Enrichment and persistence of a dominant multisegment DVG in Stock 4. (A) Stock 4 was enriched in multisegment recombination events relative to earlier samples (see Figure 1), with a multisegment-to-deletion read ratio of approximately 1:3 (junction reads per million ×), in contrast to 1:12 in Figure 1. (B) RT-PCR validation of the S4-2 DVG multisegment recombination event between segments 4 and 2 using primers flanking the predicted junction site. The amplicon was detected in passage 0 (P0), passage 1 (P1), and passage 2 (P2), but not in the negative control of an IAV PR8 stock known to not contain S4-2 DVG (via Illumina sequencing). Ladder: GeneRuler 100 bp DNA ladder.