Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nobusawa, E. Articles by Sato, K. Search for Related Content PubMed PubMed Citation Articles by Nobusawa, E. Articles by Sato, K.

 Previous Article  |  Next Article 

Journal of Virology, April 2006, p. 3675-3678, Vol. 80, No. 7
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.7.3675-3678.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Eri Nobusawa^1* and Katsuhiko Sato²

Department of Microbiology and Infection, Nagoya City University Graduate School of Medical Science,¹ Department of Microbiology, Aichi Prefectural Institute of Public Health, Nagoya City, Japan²

Received 28 September 2005/ Accepted 13 January 2006

Top Abstract Text References

 
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 x 10^–6 and 0.6 x 10^–6 mutations per site per infectious cycle, which, when extended to 1 year, agree well with the published annual evolutionary rates.

Top Abstract Text References

 
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 x 10^–5 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.

Since the 1991-1992 and 1992-1993 influenza seasons, the receptor-binding specificity of the A viruses, both H1N1 and H3N2, has changed (19, 20, 21). In particular, the binding affinity of H3N2 viruses to MDCK cells has become weaker (22). To avoid this effect, we have used viruses isolated before this change occurred. All viruses used in this study, A/Aichi/1/87(H1N1) (A/1/87), A/Aichi/12/92(H3N2) (A/12/92), B/Aichi/29/99 (B/29/99), and B/Aichi/44/01 (B/44/01), were directly isolated from clinical samples in MDCK cells.

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.

View larger version (34K): [in this window] [in a new window]

FIG. 1. Measuring the mutation rate during the growth of a single plaque. Plaque formation was performed in confluent monolayers of MDCK cells infected with A and B viruses. A well-isolated parent plaque of approximately 2 mm in diameter was picked at an appropriate time postinfection. 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 µl of minimal essential medium containing 0.2% bovine serum albumin, made into aliquots, and stored in a deep freezer (–80°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.

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.

View this table: [in this window] [in a new window]

TABLE 1. Analysis of mutation rate for NS genes of human influenza viruses

View this table: [in this window] [in a new window]

TABLE 2. Mutations observed in NS genes of descendant plaque viruses compared to the parent plaque

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 nucleotides, giving an observed mutation frequency for BNS of (2 ± 2)/461,304 = 0.4 x 10^–5 ± 0.3 x 10^–5 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 ± 5)/352,359 = 1.4 x 10^–5 ± 0.6 x 10^–5 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 x 10^–5 mutations/site/7 infectious cycles = 2.0 x 10^–6 mutations/site/infectious cycle, and 0.4 x 10^–5 mutations/site/7 infectious cycles = 0.6 x 10^–6 mutations/site/infectious cycle, respectively.

View larger version (18K): [in this window] [in a new window]

FIG. 2. Comparison of the generation times of A and B viruses in MDCK cells. Confluent monolayers of MDCK cells (5 x 105 cells) were infected with A/Aichi/1/87 and B/Aichi/29/99 at a multiplicity of infection of 8, kept at room temperature for 1 h, washed three times with minimal essential medium, and then incubated in 500 µl of overlay medium (Table 1, footnote a) excluding agar and DEAE-dextran, at 34 or 37°C. At 0 and 5 h postinfection and at each subsequent hour, a sample of the supernatant was removed and analyzed by plaque titration in MDCK cells. The PFU per 500 µl are shown versus the hours postinfection. (Inset) The generation times were determined as the point of maximum release of PFU per hour in each one-step growth experiment (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.

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 – ANS) = –(1.4 ± 1.0) x 10^–6 mutations/site/infectious cycle, giving a z-score of –1.4 and P = 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. Since the parent plaques for the A and B viruses were grown for 48 and 71 h, and since a year is 8,760 h long, if the viruses propagated continuously all through the year, we estimate evolutionary rates for the A and B viruses of 1.4 x 10^–5 mutations/site/48 h x 8,760 h = 2.6 x 10^–3 mutations/site/year and 0.4 x 10^–5 mutations/site/71 h x 8,760 h = 0.5 x 10^–3 mutations/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 x 10^–3 to 2.2 x 10^–3 and 0.45 x 10^–3 to 0.8 x 10^–3 nucleotide substitutions/site/year, respectively, on the basis 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.

 
* Corresponding author. Mailing address: Department of Microbiology and Infection, Nagoya City University Graduate School of Medical Science, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya City 467-8601, Japan. Phone: 81-52-853-8190. Fax: 81-52-853-3638. E-mail:nobusawa{at}med.nagoya-cu.ac.jp.

Top Abstract Text References

 

1
1. Adams, M. H., and F. E. Wassermann. 1956. Frequency distribution of phage release in the one-step growth experiment. Virology 2:96-108.[CrossRef][Medline]
2
2. Air, G. M., A. J. Gibbs, W. G. Laver, and R. G. Webster. 1990. Evolutionary changes in influenza B are not primarily governed by antibody selection. Proc. Natl. Acad. Sci. USA 87:3884-3888. (Erratum, 87:6007.)[Abstract/Free Full Text]
3
3. Buonagurio, D. A., S. Nakada, J. D. Parvin, M. Krystal, P. Palese, and W. M. Fitch. 1986. Evolution of human influenza A viruses over 50 years: rapid, uniform rate of change in NS gene. Science 232:980-982.[Abstract/Free Full Text]
4
4. Cox, N. J., and C. A. Bender. 1995. The molecular epidemiology of influenza viruses. Semin. Virol. 6:359-370.
5
5. Drake, J. W., and J. J. Holland. 1999. Mutation rates among RNA viruses. Proc. Natl. Acad. Sci. USA 96:13910-13913.[Abstract/Free Full Text]
6
6. Elena, S. F., and A. Moya. 1999. Rate of deleterious mutation and the distribution of its effects on fitness in vesicular stomatitis virus. J. Evol. Biol. 12:1078-1088.[CrossRef]
7
7. Ellis, E. L., and M. Delbrück. 1939. The growth of bacteriophages. J. Gen. Physiol. 22:365-384.[Abstract/Free Full Text]
8
8. Fitch, W. M., J. M. Leiter, X. Q. Li, and P. Palese. 1991. Positive Darwinian evolution in human influenza A viruses. Proc. Natl. Acad. Sci. USA 88:4270-4274.[Abstract/Free Full Text]
9
9. Francis, T., Jr., and H. E. Moore. 1940. A study of the neurotropic tendency in strains of the virus of epidemic influenza. J. Exp. Med. 72:717-728.[Abstract]
10
10. Green, P. M., and F. Giannelli. 1994. Direct sequencing of PCR-amplified DNA. Mol. Biotechnol. 1:117-124.[Medline]
11
11. Hiromoto, Y., T. Saito, S. E. Lindstrom, Y. Li, R. Nerome, S. Sugita, M. Shinjoh, and K. Nerome. 2000. Phylogenetic analysis of the three polymerase genes (PB1, PB2 and PA) of influenza B virus. J. Gen. Virol. 81:929-937.[Abstract/Free Full Text]
12
12. Ina, Y., and T. Gojobori. 1994. Statistical analysis of nucleotide sequences of the hemagglutinin gene of human influenza A viruses. Proc. Natl. Acad. Sci. USA 91:8388-8392.[Abstract/Free Full Text]
13
13. Kawaoka, Y., O. T. Gorman, T. Ito, K. Wells, R. O. Donis, M. R. Castrucci, I. Donatelli, and R. G. Webster. 1998. Influence of host species on the evolution of the nonstructural (NS) gene of influenza A viruses. Virus Res. 55:143-156.[CrossRef][Medline]
14
14. Kimura, M. 1991. Recent development of the neutral theory viewed from the Wrightian tradition of theoretical population genetics. Proc. Natl. Acad. Sci. USA 88:5969-5973.[Abstract/Free Full Text]
15
15. Lindstrom, S. E., Y. Hiromoto, H. Nishimura, T. Saito, R. Nerome, and K. Nerome. 1999. Comparative analysis of evolutionary mechanisms of the hemagglutinin and three internal protein genes of influenza B virus: multiple cocirculating lineages and frequent reassortment of the NP, M, and NS genes. J. Virol. 73:4413-4426.[Abstract/Free Full Text]
16
16. Lubeck, M. D., J. L. Schulman, and P. Palese. 1980. Antigenic variants of influenza viruses: marked differences in the frequencies of variants selected with different monoclonal antibodies. Virology 102:458-462.[CrossRef][Medline]
17
17. Ludwig, S., U. Schultz, J. Mandler, W. M. Fitch, and C. Scholtissek. 1991. Phylogenetic relationship of the nonstructural (NS) genes of influenza A viruses. Virology 183:566-577.[CrossRef][Medline]
18
18. Luria, S. E., and M. Delbrück. 1943. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28:491-511.[Free Full Text]
19
19. Medeiros, R., N. Escriou, N. Naffakh, J. C. Manuguerra, and S. van der Werf. 2001. Hemagglutinin residues of recent human A(H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes. Virology 289:74-85.[CrossRef][Medline]
20
20. Morishita, T., E. Nobusawa, K. Nakajima, and S. Nakajima. 1996. Studies on the molecular basis for loss of the ability of recent influenza A (H1N1) virus strains to agglutinate chicken erythrocytes. J. Gen. Virol. 77:2499-2506.[Abstract/Free Full Text]
21
21. Nobusawa, E., H. Ishihara, T. Morishita, K. Sato, and K. Nakajima. 2000. Change in receptor-binding specificity of recent human influenza A viruses (H3N2): a single amino acid change in hemagglutinin altered its recognition of sialyloligosaccharides. Virology 278:587-596.[CrossRef][Medline]
22
22. Nobusawa, E., K. Nakajima, S. Kozuka, and H. Ishihara. 2004. Change in receptor-binding specificity of recent human influenza A viruses (H3N2) affects recognition of the receptor on MDCK cells, p. 472-475. In Y. Kawaoka (ed.), Options for the control of influenza V. Elsevier, Amsterdam, The Netherlands.
23
23. Parvin, J. D., A. Moscona, W. T. Pan, J. M. Leider, and P. Palese. 1986. Measurement of the mutation rates of animal viruses: influenza A virus and poliovirus type 1. J. Virol. 59:377-383.[Abstract/Free Full Text]
24
24. Rao, V. B. 1994. Direct sequencing of polymerase chain reaction-amplified DNA. Anal. Biochem. 216:1-14.[CrossRef][Medline]
25
25. Stech, J., X. Xiong, C. Scholtissek, and R. G. Webster. 1999. Independence of evolutionary and mutational rates after transmission of avian influenza viruses to swine. J. Virol. 73:1878-1884.[Abstract/Free Full Text]
26
26. Suarez, P., J. Valcarcel, and J. Ortin. 1992. Heterogeneity of the mutation rates of influenza A viruses: isolation of mutator mutants. J. Virol. 66:2491-2494.[Abstract/Free Full Text]
27
27. Yamashita, M., M. Krystal, W. Fitch, and P. Palese. 1988. Influenza B virus evolution: cocirculating lineages and comparison of evolutionary pattern with those of influenza A and C viruses. Virology 163:112-122.[CrossRef][Medline]

---

Journal of Virology, April 2006, p. 3675-3678, Vol. 80, No. 7
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.7.3675-3678.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Hai, R., Martinez-Sobrido, L., Fraser, K. A., Ayllon, J., Garcia-Sastre, A., Palese, P. (2008). Influenza B Virus NS1-Truncated Mutants: Live-Attenuated Vaccine Approach. J. Virol. 82: 10580-10590 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nobusawa, E. Articles by Sato, K. Search for Related Content PubMed PubMed Citation Articles by Nobusawa, E. Articles by Sato, K.