Re: Swine Flu May Merge With Other Flu Viruses, CDC Says
Homologous recombination evidence in human and swine influenza A viruses
Cheng-Qiang Hea, , , Guan-Zhu Hana, Dong Wanga, Wei Liuc, Guo-Rong Lia, Xi-Ping Liua and Nai-Zheng Dingb, ,
aCollege of Life Science, Shandong Normal University, Shandong Province, Jinan 250014, China bCollege of Life Science, Central South University, Changsha 410012, China cLaboratory of Molecular Biology, Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China
Received 14 March 2008;
revised 6 May 2008;
accepted 15 July 2008.
Available online 21 August 2008.
Abstract
Dynamic gene mutation and the reassortment of genes have been considered as the key factors responsible for influenza A virus virulence and host tropism change. This study reports several significant evidence demonstrating that homologous recombination also takes place between influenza A viruses in human and swine lineages. Moreover, in a mosaic descended from swine H1N1 subtype and human H2N2, we found that its minor putative parent might be a derivative from the human cold-adapted vaccine lineage, which suggests that live vaccine is capable of playing a role in genetic change of influenza A virus via recombination with circulating viruses. These results would be important for knowing the molecular mechanism of mammal influenza A virus heredity and evolution.
Keywords: Influenza A virus; Homologous recombination; Vaccine
Article Outline
Introduction
Results and discussion
A/swine/Ontario/57561/03(H1N1)
A/swine/Ontario/53518/03(H1N1)
Methods
Acknowledgements
Appendix A. Supplementary data
References
Introduction
Influenza viruses threaten significantly both human and animal health. In the last hundred years, several influenza pandemics were responsible for the deaths of at least 40 million people (Horimoto and Kawaoka, 2005). Recently, a highly pathogenic avian influenza virus (AIV; H5N1) has resulted in the death of more than 200 people and the slaughter of millions of poultry in Asia, Europe and Africa, raising concern over the possibility of a new influenza pandemic among the world's immunologically naive populations (Horimoto and Kawaoka, 2005).
The virus belongs to the Orthomyxoviridae family and contains eight segments of single-stranded RNA (ssRNA) which encode 11 proteins, PB2, PB1, PB1-F2, PA, HA, NP, NA, M1, M2, NS1 and NS2 (Nelson and Holmes, 2007). These segments allow for the swapping and exchange of gene segments between different strains. Specifically it occurs when the human influenza viruses swap their HA glycoprotein, NA glycoprotein or polymerase (PB1, PB2, PA) segments with those of avian and pig Influenza A viruses. Although the origin of the H1N1 strain that caused the severe pandemic of 1918 is less clear and the source of much debate ([Antonovics et al., 2006], [Gibbs and Gibbs, 2006] and [Taubenberger et al., 2005]), reassortment among HA and NA subtypes was fundamental in the human pandemics of 1957 (H2N2 subtype) and 1968 (H3N2 subtype), which also acquired a new basic polymerase 1 (PB1) segment (Lindstrom et al., 2004).
A key molecular basis that drives the virulence change of a virus is amino acid mutation. Dynamic gene mutant has been shown to play an important role in the virulence change of AIV ([Hatta et al., 2001] and [Hulse-Post et al., 2007]), but homologous recombination also plays an important role in the evolution of some RNA viruses ([Kirkegaard and Baltimore, 1986], [Lai, 1992] and [Nagy and Simon, 1997]). Virulent variants of some other viruses have been generated by homologous recombination ([Anderson et al., 2000], [Kew et al., 2002], [Pita et al., 2001] and [Worobey et al., 1999]). For influenza A virus, there has been ample evidence that influenza viruses undergo various forms of non-homologous recombination. For example, a recombination can occur between HA and nucleoprotein gene (Orlich et al., 1994). Similarly, increased viral pathogenicity after insertion of a 28S ribosomal RNA sequence into the haemagglutinin gene of an influenza virus was also found (Khatchikian et al., 1989). However, the occurrence of homologous recombination within segments is far from proven (Nelson and Holmes, 2007).
In order to clarify whether homologous recombination drives the evolution of human influenza A, we analyzed the H1N1 subtype of about 3800 genes and found several significant evidences of recombination between swine H1N1 and human H2N2 subtypes in PA, between swine lineage strains in PB2, and between human lineage strains in HA and NP. Particularly, in a mosaic PA of swine H1N1, one putative parent seems to be derived from a cold-adapted avirulent vaccine lineage of human H2N2. These data show that the intragenic recombination can indeed play a role in driving the evolution of influenza A virus. In addition, the avirulent vaccine could shape the evolution of the virus via homologous recombination with circulating influenza A virus.
Results and discussion
We performed different segment comparisons of available H1N1 subtype sequences (total 3815 complete gene segment sequences, HA, 485; NA, 472; PA, 459; PB1, 467; PB2, 457; M, 478; NS, 476; NP, 521) to find evidence of recombination between influenza A and to identify potential breakpoints for any such events. Among 3815 complete genes of influenza A, at least four mosaics were found (Table 1). In particular, the natural recombinant A/swine/Ontario/57561/03(H1N1) (DQ280191) is descended from swine H1N1 and human H2N2 in the gene PA. The results show that the homologous recombination can drive mammal influenza A virus evolution, and might result in potential change of virus virulence and host tropism.
Table 1.
Influenza A virus strains with significant evidence for recombination
No. Accession no. Mosaic virus strain Gene Putative parent lineages Breakpoint(s) pSH
1 DQ280191 A/swine/Ontario/57561/03(H1N1) PA A/swine/Ontario/55383/04(H1N2) 515 < 0.001
A/Ann Arbor/6/1960(H2N2)
2 DQ280213 A/Swine/Ontario/53518/03 PB2 A/Swine/Korea/CY02/02(H1N2) 538 < 0.001
A/swine/Alberta/56626/03(H1N1)
3 DQ415318 A/Taiwan/4845/99(H1N1) HA A/WSN/33(H1N1) 136, 623, 960 < 0.001
A/Wellington/24/2000(H1N1)
4 AF255749 A/Hong Kong/498/97(H3N2) NP A/Hong Kong/427/98(H1N1) 223 < 0.01
A/Hong Kong/497/97(H3N2)
Full-size table
Note. pSH indicates probability of Shimodaira?Hasegawa test.
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A/swine/Ontario/57561/03(H1N1)
Recently, Karasin et al. (2006) reported three novel genotypes of H1 influenza viruses recovered from pigs in Ontario of Canada. Reassortment between influenza viruses was found to be the cause resulting in these novel genotypes (Karasin et al., 2006). These viruses were isolated from pigs that ranged in age from 3-week-old nursery piglets to adult sows and that exhibited various clinical signs typical of swine influenza illness (Karasin et al., 2006). They were passaged no more than once beyond initial isolation before sequencing (Karasin et al., 2006). Their sequences were checked multiple times and determined using multiple primer pairs to be sure that none of the reported sequences were artifacts (personal communication to Prof. Olsen CW). Among these viruses, A/swine/Ontario/57561/03(H1N1) and A/swine/Ontario/53518/03(H1N1) were suspected as intragenic recombinants in PA and PB2 segment respectively.
We conducted a similarity analysis using A/swine/Ontario/57561/03(H1N1) as a query. The standard similarity plot, constructed using all sites, reveals that the PA sequence exhibits greater affinity with the influenza A human lineage than the swine lineage in the region from position 1 to 548 (Fig. 1A). On the contrary, the mosaic shares higher sequence similarity with the swine lineage than the human lineage in the region from 549 to 2147. The incongruent phylogenetic trees incorporating the avian lineage provide a robust, informative test of the recombination hypothesis (Worobey et al., 2002). When the maximum likelihood trees separated by position 548 were constructed, a significant discrepancy (Shimodaira?Hasegawa test, p < 0.001) between phylogenetic trees constitutes a powerful evidence for recombination in A/swine/Ontario/57561/03(H1N1) (Figs. 1C and D).
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Fig. 1. The evidences for recombination in PA gene of the strain A/swine/Ontario/57561/03(H1N1). A, PA similarity from SimPlot analysis of A/swine/Ontario/57561/03(H1N1) is shown. Comparison of influenza A strain A/swine/Ontario/57561/03(H1N1) with the swine and human influenza A strains. The y-axis gives the percentage of similarity within a sliding window of 200 bp wide centered on the position plotted, with a step size between plots of 20 bp. B?D, phylogenetic trees for PA genes. The relationships of nucleotide sequences of the PA gene derived from humans, pigs and birds are indicated by an unrooted tree constructed using the ML method, implemented in the Phyml as described in Methods. The values of bootstrap are shown below or above the branch, respectively. The scale corresponds to the number of nucleotide substitutions per site in all trees. Only bootstrap values more than 70% are shown on each branch. The GenBank number of each segment is also listed after its name. B, the phylogenetic tree of complete PA ORF. C, the phylogenetic tree of the region from position 1 to 548. D, the phylogenetic tree of the region from position 549 to 2147. E?H, identification of recombination breakpoints in detail. E, similarity plot as in Fig. 1A with a window size of 200 bp and a step size of 20 bp. Vertical lines indicate the breakpoint identified by maximization of χ2 and sample Akaike Information Criterion as described in Methods. Comparison of the strain A/swine/Ontario/57561/03(H1N1) with putative parents A/Ann Arbor/6/60 and A/swine/Ontario/55383/04(H1N2) is shown in E. The rest is the same as Fig. 1A. F, the result of Bootscaning. The y-axis gives the percentage of permutated trees using a sliding window of 200 bp wide centered on the position plotted, with a step size between plots of 20 bp. DE-R49/99 was used as the outgroup to determine the breakpoints. G and H are the ML phylogenies inferred for the 2 regions delimited by the informative-site analysis as described in Methods.
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Because two cold-adapted avirulent H2N2 vaccines, A/Ann Arbor/6/1960(H2N2) and A/Leningrad/134/47/57(H2N2) were not included in our data initially, we were puzzled when its putative minor parent of the mosaic virus was identified. We found the region from nucleotide 1 to 548 of the mosaic strain fell into the subgroup of H2N2 isolated from 1957 to 1968 in phylogenetic tree. However, the mosaic strain was isolated in 2003. PA gene of human H2N2 was estimated to have evolved at rates of 2.49 ? 10 − 3 ns/s/year (R2 = 0.92) (Lindstrom et al., 2004). The sequence similarity between the mosaic isolated in 2003 and the H2N2 group isolated in 1960s should be about 90%. However, we found that the mosaic shared nearly 99% sequence similarity with some strains isolated in 1968. It would suggest that the putative parent of the mosaic had nearly no evolution during the 45 years when it circulated in the field, and this could not be true. A hypothesis would be that the minor parent has been descended from an avirulent vaccine of H2N2. In agreement, when the two vaccines A/Ann Arbor/6/1960(H2N2) and A/Leningrad/134/47/57(H2N2) were incorporated into the phylogenetic trees, the mosaic strain came forth in the lineage of H2N2 vaccines (Fig. 1C). The avirulent vaccine A/Ann Arbor/6/60 shares the highest sequence similarity (99.45%, 543/546) with the mosaic strain from position 1 to 546 among all isolates deposited in the GenBank. A CAIV-T (tradename FluMistTM, Aviron, Mt. View, CA) derived from A/Ann Arbor/6/60 ([Gruber, 2002] and [Herlocher et al., 1993]) has been licensed in the US since 2003, suggesting that it might be shed by the vaccinees in the Ontario region and act as the putative minor parent of the mosaic. Therefore, we proposed that the putative minor parent of the mosaic might have descended from the lineage of the avirulent vaccine of human H2N2. This finding also suggested that the avirulent vaccine of human influenza A was able to shape the genetic diversity of the virus via homologous recombination with circulating viruses.
After the putative parents were identified, further recombination analyses were carried out to determine the potential breakpoints. A single breakpoint was located in a parsimonious region from 511?542 with the maximization of χ2 using the program SimPlot. The most likely breakpoint was found exactly at site 515 according to Single Breakpoint Analysis in GARD (Figs. 1E?H).
A/swine/Ontario/53518/03(H1N1)
In a previous study, A/Swine/Ontario/53518/03 was found to be a human?swine reassortant in PB1 that exhibited various clinical signs typical of swine influenza illness (Karasin et al., 2006). It is very interesting that its PB2 is also an intragenic mosaic (Fig. 2).
We conducted a similarity analysis using A/Swine/Ontario/53518/03 as a query. The standard similarity plot reveals that the PB2 sequence exhibits greater affinity with the swine influenza A virus H1N1 subgroup than the H1N2 subgroup from position 1 to 537 (Fig. 1A). On the contrary, the mosaic shares higher sequence similarity with the swine H1N2 subgroup than the H1N1 subgroup from position 537 to 2273. A significant discrepancy between phylogenetic trees inferred for nucleotide sequences of each recombination region was found (Shimodaira?Hasegawa test, p < 0.001) when the maximum composite likelihood trees were constructed incorporating the human lineage (Figs. 2B?D). These results constitute powerful evidences for PB2 mosaic in A/swine/Ontario/57561/03(H1N1) descended from swine H1N1 and H1N2 influenza A virus.
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Fig. 2. The evidences for recombination in PB2 gene of the strain A/swine/Ontario/53518/03(H1N1). A, comparison of PB2 of influenza A virus strain A/swine/Ontario/53518/03(H1N1) with all swine influenza A strains listed in Fig. 2B. The y-axis gives the percentage of identity within a sliding window of 200 bp wide centered on the position plotted, with a step size between plots of 20 bp. B, ML phylogenetic tree for full-length open reading frame (ORF) of PB2 incorporating the human lineage. The tree was constructed as described in Methods. And the rest is the same as Fig.1B. C, the phylogenetic tree of the region from position 1 to 538. D, the phylogenetic tree of the region from position 539 to 2273. E, sequence similarity comparison of the mosaic strain with its putative parents, A/swine/Alberta/56626/03(H1N1) and A/Swine/Korea/CY02/02(H1N2). The red vertical line shows the recombination breakpoints with the maximization of χ2. F, bootscanning of PB2 of the mosaic and its parents. The y-axis gives the percentage of permutated trees using a sliding window of 200 bp wide centered on the position plotted, with a step size between plots of 20 bp. The strain A/Memphis/3/1983(H1N1) was used as outgroup. The rest is the same as E.
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To find the putative parents of A/Swine/Ontario/53518/03, we performed BLAST using PB2 of A/Swine/Ontario/53518/03 as a query in the GenBank and found that A/swine/Alberta/56626/03 shared the highest sequence similarity with the mosaic gene from position 1 (initial code) to 537. Interestingly, an isolate from Korea A/Swine/Korea/CY02/02 shared the highest sequence similarity with the mosaic from nt 537 to 2273. This suggested the existence of a global reservoir of influenza A virus. Therefore, we proposed that the two strains might be the putative parents of the mosaic. The comparisons between the mosaic and its putative parents are shown in Figs. 2E and F. A breakpoint, from position 537 to 540 was found in PB2 gene of A/swine/Ontario/53518/03(H1N1) by maximization of χ2 and Findsites sub-program of Simplot Program. And running the GARD online, the exact breakpoint was located in position 538. The mosaic PB2 shares 100% sequence similarity with A/swine/Alberta/56626/03 (versus 84.34% with A/Swine/Korea/CY02/02) from nt 1 to 538. However, from nt 539 to 2237, it also shares 99.89% sequence similarity with A/Swine/Korea/CY02/02 (versus 82.86% with A/swine/Alberta/56626/03). These analyses determined further that the PB2 of A/swine/Ontario/53518/03 is an intragenic recombinant.
In this study, we analyzed 3815 segment sequences of mammalian influenza A virus (equal to about 440 complete genomes) and only found 4 mosaic strains (Table 1). It means that the frequency of intragenic recombination is not high in mammalian influenza A virus. However, intragenic recombination happens indeed between different or same subtype viruses; although the recombination has always been ignored in influenza A virus. Recombination, like point mutation and reassortment, can also produce novel virus variants and results in increased virulence ([Anderson et al., 2000], [Kew et al., 2002] and [Worobey et al., 1999]). Here, we find that recombination can occur in PB2 and PA, HA (between human H1N1 subtype strains) and NP (between human H1N1 subtype strain and H3N2 subtype strain) (Table 1). The virulence of influenza A viruses is associated with their HA ([Horimoto and Kawaoka, 1994], [Perdue et al., 1997] and [Senne et al., 1996]). Mutations in the HA gene have produced highly pathogenic strains, and the major pandemics of 1957 and 1968 might be largely caused by the introduction of antigenically novel HA genes from bird-infecting influenzas ([Khatchikian et al., 1989] and [Subbarao et al., 1998]). Additionally, the polymerase complex genes were also found to contribute to the high virulence of the human H5N1 influenza virus (Salomon et al., 2006). And PB2 mutation is responsible for the virulence change in AIV (Hatta et al., 2001). If recombination happens in genes responsible for virulence and host tropism, its potential effect on the change of virulence and host tropism might be fatal.
Reassortment and recombination processes will allow some viruses to acquire many of the key adaptive mutations in a single step and hence make a major leap in fitness space (Kuiken et al., 2006), which might result in a change of host tropism of the virus. A reassortment of gene segments between pig and human influenza virus has occurred creating an entirely novel Influenza A virus strain capable of infecting humans (Steinhauer and Skehel, 2002), and might result in a population that is entirely immunologically naive to these novel viruses in human being (Russell and Webster, 2005). Similarly, the intragenic recombination between influenza virus swine lineage and human lineage is also capable of creating a novel swine lineage virus readily adaptable to human hosts. Especially, the recombination occurring in polymerase complex between human and swine influenza A virus might more easily result in a novel virus adapting to human since the viral heterotrimeric polymerase complex is considered having a role in host specificity ([Gabriel et al., 2005] and [Taubenberger et al., 2005]). Therefore, the possibility of a recombination event triggering a pandemic by altering gene structure or function and/or by permitting the virus to switch hosts from some other mammal into humans should not to be neglected.
Live vaccine derived from an A/Leningrad/134/47/57 (H2N2) master strain, a cold-adapted H2N2 strain (Rudenko et al., 1993), has been used in Russia and other countries. Avirulent vaccine CAIV-T (tradename FluMistTM, Aviron, Mt. View, CA) derived from A/Ann Arbor/6/60 has been licensed in the US. The advent of live attenuated cold-adapted influenza virus vaccine (CAIV-T) administered intranasally may provide a convenient and effective alternative approach for influenza immunization (Gruber, 2002). However, since live vaccine strains are potentially released into the environment by vaccinees, safety issues concerning medical as well as environmental aspects must be considered. An important aspect concerns the exchange of genetic information between the vaccine of interest and another vaccine or wild-type strains of the carrier organism. Here, we found the natural recombinant which might be derived from a live vaccine strain. It suggests that live avirulent vaccine can play some and perhaps a very important role in speeding up the evolution of influenza A virus. It has been reported that a mosaic bovine viral diarrhea virus between a persisting pestivirus and a vaccine strain was a cytopathogenic virus that induced lethal diseases (Becher et al., 2001). And there have been recent polio epidemics in Hispaniola that arose from similar recombinants involving vaccine virus and other endogenous enteroviruses, which threatens the entire WHO polio eradication program (Kew et al., 2002). Therefore, it might be necessary to evaluate the effect of various vaccine recombination on influenza A virus virulence and host tropism. From an environmental perspective, inactivated influenza A vaccine should be safer.
In conclusion, our study provided powerful evidence that intragenic recombination occurs in mammalian influenza A viruses from different species, which shows that homologous recombination may play a role in driving the evolution of mammalian influenza A viruses and has the potential to change their virulence and host tropism. Finally, our results suggest that live vaccine is capable of speeding up viral evolution via recombination with circulating viruses.
Methods
We collected all the H1N1 subtype genes (total 3815 complete gene sequences, HA, 485; NA, 472; PA, 459; PB1, 467; PB2, 457; M, 478; NS, 476; NP, 521) that are deposited in the GenBank and analyzed their homologous recombination in the study. Multialignment was finished by using CLUSTALW (Thompson et al., 2003). Gaps were removed before further analyses were carried out. The neighbor-joining (NJ) phylogenetic tree was generated using the maximum composite likelihood (MCL) model implementing Mega4 (Tamura et al., 2007). Maximum Likelihood trees were also constructed to determine the recombination events using Phyml online tool (Guindon and Gascuel, 2003) (
http://atgc.lirmm.fr/phyml/). The trees were tested using bootstrap (1000 replications). Putative recombinant sequences were identified with the SimPlot program (Lole et al., 1999). Shimodaira?Hasegawa test was implemented to prove whether phylogenetic trees estimated from different regions are significantly different employing Treetest program (
http://aix1.uottawa.ca/sarisbro/). The putative parents were searched using putative mosaic as a query by BLAST in the GenBank. Recombination breakpoints were analyzed by maximization of χ2 using SimPlot combined with Akaike Information Criterion (AIC) (AICc) (Kosakovsky Pond et al., 2006). The recombination events were also further analyzed using GARD (Genetic Algorithm Recombination Detection) online (
http://www.datamonkey.org/GARD/) (Kosakovsky Pond et al., 2006).
Acknowledgments
We would like to thank Prof. Chris Olsen (University of Wisconsin?Madison) for the important information of the two isolates A/swine/Ontario/57561/03(H1N1) and A/swine/Ontario/53518/03(H1N1).
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Appendix A. Supplementary data
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