Comparison of the Mutation Rates of Human Influenza A and B Viruses

ABSTRACT Human influenza A viruses evolve more rapidly than influenza B viruses. To clarify the cause of this difference, we have evaluated the mutation rate of the nonstructural gene as revealed by the genetic diversity observed during the growth of individual plaques in MDCK cells. Six plaques were studied, representing two strains each of type A and B viruses. A total of 813,663 nucleotides were sequenced, giving rates of 2.0 × 10−6 and 0.6 × 10−6 mutations per site per infectious cycle, which, when extended to 1 year, agree well with the published annual evolutionary rates.

Human influenza A and B viruses (A and B viruses) are equally prevalent among human beings, yet most A virus genes evolve two to three times faster than the corresponding genes in B viruses (11,13,15). The high evolutionary rate of A viruses has been attributed to positive selection by the human immune system (2-4, 8, 12), and the difference between A and B to an absence of such selection in B viruses (2,27). However, a possible alternative explanation of the difference in evolutionary rates, a difference in mutation rates at the molecular level, has not been investigated (27). To correct this lack, we have compared the mutation rates of the nonstructural (NS) genes of several circulating A and B viruses during replication in Madin-Darby canine kidney (MDCK) cells, which are unaffected by the human immune system.
Previously, Parvin et al. reported a mutation rate for the NS gene of influenza A virus A/WSN/33(H1N1) (WSN) of 1.5 ϫ 10 Ϫ5 mutations per nucleotide per infectious cycle during the growth of a single plaque in MDCK cells (23). In their study, Parvin et al. demonstrated that most of the NS mutants they detected replicated with fitness similar to that of the parent virus. This suggests that the MDCK cells did not encourage or discourage the appearance of these mutations, and hence are a suitable medium for the study of mutation rates. We have adopted their method (23), with some modifications.
The mutation rates for the NS genes of A (ANS) and B (BNS) viruses were measured using the procedure illustrated in Fig. 1. In brief, well-isolated parent plaques were picked when they had reached a diameter of approximately 2 mm at 48 and 71 h postinfection for A and B viruses, respectively. The parent plaques were used to produce a series of descendant plaques grown to similar diameters.
The nucleotide sequences of the NS genes in each plaque were determined directly using the reverse transcription-PCR (RT-PCR) technique (Table 1) (21). Mutations were identified by comparing the sequence in randomly selected descendant plaques to the sequence in the corresponding parent plaque using sequence analysis software, GENETYX-MAC ver.11.0 (Genetyx Corporation, Japan). The direct sequencing of RT-PCR products produces an average sequence of many independent copies of the viral cDNA. Although some molecules will be miscopied during the PCR process, they represent a small fraction of the total and do not affect the average sequence (10,24). All mutations ( Table 2) were confirmed by sequencing with the different sense primers listed in Table 1 a total of at least three times.
Most experiments counting the frequency of mutants in viral plaques find a high variability (16,25,26). Most of this variability arises from replication errors occurring early in the growth of the plaque. Such random events, when they occur, are amplified to produce large clone lines of identical mutants ( Fig. 1), greatly increasing the variance of the number of mutants detected (18,25). With direct sequencing, it is possible to distinguish each replication error. Counts of the number of unique mutant sequences are related to the number of mutation events, as opposed to the number of progeny resulting from them. Such counts vary only as expected from Poisson statistics. Although no mutant clone lines were detected among the samples taken during the main experiment, in a preliminary test with a B/44/01 parent plaque grown for 96 h, a sample of 169 descendants revealed three mutants. Of these, two exhibited the same mutant sequence, indicating they were very likely clones descended from the same mutation event. By contrast, an A/12/92 parent plaque grown for 72 h contained three different mutant sequences in a sample of 162 descendants.
The results from the main sequencing experiment are listed in Tables 1 and 2. A total of six experiments were performed, three with B viruses (Table 1, experiments 1 to 3) and three with A viruses (Table 1, experiments 4 to 6). For B viruses, a total of two mutations were detected among 461,304 nucleo-tides, giving an observed mutation frequency for BNS of (2 Ϯ ͌2)/461,304 ϭ 0.4 ϫ 10 Ϫ5 Ϯ 0.3 ϫ 10 Ϫ5 mutations/site. For A viruses, the first two experiments with primary plaques formed at 34°C (Table 1, experiments 4 and 5) indicated a mutation frequency far lower than that reported by Parvin et al. (23), who grew their parent plaque at 37°C. To examine the possible effect of temperature on mutation frequency, we remeasured A/1/87 at 37°C. The number of mutations detected was identical to that at 34°C. Taking all three A virus experiments together, the average mutation frequency for ANS in the parent plaques was (5 Ϯ ͌5)/352,359 ϭ 1.4 ϫ 10 Ϫ5 Ϯ 0.6 ϫ 10 Ϫ5 mutations/site.
The higher mutation frequency of WSN(H1N1) compared to A/1/87(H1N1) may reflect the peculiar character of WSN, a neurotropic virus, produced by repeated pulmonary and intracerebral infection of mice (9). There may also have been some problem with the technology available in 1986. In the absence of PCR and simplified sequence technology, Parvin et al. (23) were unable to repeat the sequence analysis to confirm mutations by reading both strands of the cDNA. For this reason, their reported number of mutations might not be accurate.
To compare the mutation rate per infectious cycle of the A and B viruses, we determined the generation time of each virus in MDCK cells. As shown in Fig. 2, the mean generation time, when the PFU reach 50% of the final value (1,7), was about 7 and 10 hours for A and B viruses, respectively. As the dynamics of plaque growth are complicated and the budding of progeny viruses could not be synchronized, these generation times were adopted as probable values during plaque formation. Since the 2-mm-diameter parent plaques were picked at 48 h (A viruses) and 71 h (B viruses) postinfection, they correspond to approximately seven infectious cycles in both cases. Using these values, average mutation rates for ANS and BNS were calculated as 1.4 ϫ 10 Ϫ5 mutations/site/7 infectious cycles ϭ 2.0 ϫ 10 Ϫ6 mutations/site/infectious cycle, and 0.4 ϫ 10 Ϫ5 mutations/site/7 infectious cycles ϭ 0.6 ϫ 10 Ϫ6 mutations/site/infectious cycle, respectively.
It should be noted that some authors interpret "infectious" and "replication" cycle differently (5,6,23,25,26). The observed difference in mutation rates is (BNS Ϫ ANS) ϭ Ϫ(1.4 Ϯ 1.0) ϫ 10 Ϫ6 mutations/site/infectious cycle, giving a z-score of Ϫ1.4 and P ϭ 0.08. This difference in mutation rate presumably reflects a more accurate polymerase in the B virus than in the A virus.
Finally, we attempted to estimate evolutionary rates from the observed in vitro mutation rates in MDCK cells. To clone selected individual descendant viruses in the parent plaque, a second plaque formation was performed using the virus yield directly from the parent plaque. All plaque viruses were eluted from the agar plug into 300 l of minimal essential medium containing 0.2% bovine serum albumin, made into aliquots, and stored in a deep freezer (Ϫ80°C) until used. The harvested viruses were used without further modification for sequential analysis of their NS genes, which reveals the sequence of the major virus in the plaque. The magnified views of the plaques show the viruses that exist within them. Star, viruses carrying NS gene sequences identical to that of the major virus in the parent plaque; triangle, diamond, spade, club, heart, and square, viruses carrying NS gene sequences different from that of the major virus in the parent plaque. The two triangles in the parent plaque represent a clone line. One of the descendant plaques is descended from a mutant of this type.
site/year, respectively. Evolutionary rates for the NS genes of human influenza A and B viruses have been reported in the range of 1.8 ϫ 10 Ϫ3 to 2.2 ϫ 10 Ϫ3 and 0.45 ϫ 10 Ϫ3 to 0.8 ϫ 10 Ϫ3 nucleotide substitutions/site/year, respectively, on the ba- a To avoid a change in character of the viruses by adaptation to MDCK cells, we limited the number of virus amplifications in MDCK cells, prior to the experiment, to five. The overlay for plaquing consisted of minimal essential medium with 0.6% agar (Difco), 0.25% bovine serum albumin, 0.01% DEAE-dextran, 0.1% glucose, and 2.0 g of acetyl trypsin per milliliter. Viruses were plaque purified twice in MDCK cells before preparing parent plaques. Plaque formation was performed in MDCK cells at 34°C unless otherwise noted. Parent plaques were grown for 48 h for A viruses and 71 h for B viruses.
b For the nucleotide sequence analysis, the viral RNA was directly extracted from 100 l of each descendant plaque virus (300 l eluant) using Isogen LS (Nippon Gene, Japan). Half of this viral RNA was used to synthesize cDNA of the NS genes by RT-PCR as described previously (21)  c Number of nucleotide differences between descendant and parent NS genes. Details of each mutation are given in Table 2. d Ratio of number of nucleotide changes detected to number of nucleotides analyzed. e Following Parvin et al. (23), the mutation rate was determined by dividing the observed frequency of nucleotide mutations per site by the number of infectious cycles. This assumes that all mutants continue to replicate as the plaque grows. If mutant lines die out, the mutation rate obtained in this way may underestimate the true mutation rate. We are assuming seven infectious cycles for A and B viruses.    (1,7). This is generally equivalent to the point at which the PFU reaches 50% of its final value, estimated here by averaging the total PFU at 11, 12, and 14 h. The width of the distribution about the mean was likewise estimated by noting the points at which the PFU reached 16 and 84% of its final value, corresponding to plus and minus 1 standard deviation (1). The change in temperature from 34 to 37°C, tested for A/1/87 only, appeared to have no significant effect on the mean generation time.

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NOTES 3677 sis of nucleotide sequencing of circulating viruses (13,15,17). Although the generation time of viruses and the selection factors on NS gene products in vivo may not be the same as those in MDCK cells, our estimated evolutionary rates are in good agreement with the reported actual rates. Whether this agreement is a consequence of the random fixation of neutral mutations (14), of some other origin, or purely an accident remains to be solved. The current study suggests that one of the reasons for the lower evolutionary rate of influenza B viruses could be a lower mutation rate.
We are greatly indebted to J. Mosher for helpful suggestions and valuable discussion. K. Nakajima and S. Itamura also provided critical discussions. Finally, we are grateful to N. Murayama for excellent technical assistance.
This work was supported in part by a Grant-in-Aid for Research in Nagoya City University.