Influenza viruses contain a segmented genome consisting of eight minus-sense RNA segments which code for 10 known viral proteins that are capable of reassortment during coinfection of a single host with two influenza viruses. Indeed, reassortment between human influenza viruses of different subtypes and between human and swine influenza viruses has been demonstrated repeatedly (3
), and it has been suggested that swine may serve as an intermediate host in which reassortment between human, swine, and avian influenza viruses may occur to give rise to new pandemic strains (3
). Also, a natural H1N2 reassortant containing the HA of recent human H1N1 viruses and the NA of recent human H3N2 viruses has been isolated from humans in China (30
). With this evidence, it may be expected that reassortment between cocirculating human influenza viruses of the same subtype occurs. Indeed, recent phylogenetic divergence of the NA gene was suggested to be the result of genetic reassortment between recent human viruses (59
). However, through evolutionary analysis of the internal NS gene, it was proposed that reassortment among cocirculating viruses appears not to occur very often and, therefore, it has been suggested that fixation of mutations in the genes coding for the internal proteins is dependent on immune pressure on the HA protein, effectively linking the evolution of the internal genes of influenza viruses to the HA gene (7
). Although this may indeed often be the case, parallel analyses of the genes coding for the surface glycoproteins and internal proteins have never been reported. A scarcity of sequence data of the internal genes of human H3N2 viruses has made it very difficult to analyze the phylogenetic patterns of these genes in a parallel manner. Also, available sequence data is of different viruses, isolated in different years and in different parts of the world, making an accurate comparison of the evolutionary patterns of these genes very difficult. The determination in this study of the phylogenetic pathways of all eight genes of 10 recent influenza viruses revealed that the gene segments coding for the internal proteins of these viruses are evolving in a more-independent manner than was previously speculated and were not linked to the evolution of the HA gene.
Although the HA proteins of four viruses isolated in the epidemic season of 1995 were almost identical, differences were observed in the PB1, PA, NP, NA, M2, and NS1 proteins which distinguished these viruses into two pairs. With the exception of the HA gene, the differences in the proteins of 1995 viruses were further supported by evolutionary analysis, indicating that the genes of 1995 isolates consistently diverged into two distinct branch clusters (95i and 95ii) with bootstrap probabilities of 95 to 100. It was, therefore, understood that considerable variability existed among cocirculating viruses in the same epidemic season which was not apparent through analysis of the HA protein alone. Also, it appeared that distinct RNA segment constellations of 95i and 95ii viruses may have been established through reassortment among cocirculating H3N2 viruses.
As summarized in Table , evolutionary patterns determined for each of the eight genes of human H3N2 viruses isolated from 1993 to 1997 indicated that reassortment between cocirculating human influenza viruses apparently occurred during this 5-year period. Two isolates of 1995, Aki95 and Toc95, contained PB2 and NP genes which were genetically more similar to those of a 1993 virus than to those of other viruses isolated in the same epidemic season. Japanese viruses of 1997 contained HA, NA, and PB2 genes that appeared to have originated from those of the previous variant of 1996, while the genes coding for the internal PB1, PA, NP, and M proteins apparently derived from earlier viruses of the 1993-1994 season. Most notably, the evolutionary patterns of the internal genes clearly demonstrated with bootstrap probabilities of 99 to 100 that the PB1 and NP genes of 1997 viruses were most similar to those of a 1994 isolate, whereas the M genes were distinct from all Japanese isolates from 1993 to 1996 and were instead most similar to that of a Chinese isolate of 1993. Even though the topology of the phylogenetic tree for the PA gene did not correlate well with the dates of the isolates, the PA genes of the 1997 isolates were shown to have diverged from a virus other than those of 1995 or 1996. Although it was reported that the NS genes of human H3N2 viruses evolves in a rapid and sequential fashion (7
), our analyses of the NS genes of recent H3N2 viruses revealed a nonlinear pattern of evolution and amino acid substitutions which seldom survived longer than one season. The nonlinear evolutionary pattern of the NS genes created difficulty in the determination of the origins of the NS genes of recent viruses, since significant probabilities for the internal branches could not be calculated. Nevertheless, the evidence suggested that distinct RNA constellations appeared to be a result of genetic recombination between cocirculating human H3N2 viruses and that genetic exchange may or may not accompany antigenic drift of the HA protein.
TABLE 4 Proposed derivative strains of RNA segments of recent human H3N2viruses
It has been suspected for some time that new epidemic variants of H3N2 influenza virus originated from China (49
), since antigenically variable viruses may circulate for some time in China before becoming epidemic. This was apparent when A/Beijing/32/92-like viruses were isolated as early as 1990 in China but did not become epidemic until 1993 (13
). A/Beijing/32/92-like viruses, therefore, appear to have circulated for at least 3 years in China before becoming the predominant epidemic strain globally. It is unclear why a particular strain will suddenly emerge after circulating for years in China, although it is generally thought that this is because the HA protein has not undergone sufficient antigenic change to effectively evade established immunity in the human population. In the five epidemic seasons in Japan investigated in this report (1993 to 1997), the relative morbidity due to H3N2 viruses in the 1993, 1995, and 1997 seasons was considerably higher than in the 1994 or 1996 seasons. High levels of morbidity due to H3N2 virus activity in 1995 and 1997 coincided with observed variability in the internal proteins, which was apparently the result of genetic reassortment. The results of this study provide evidence that strongly suggests for the first time that genetic exchange among cocirculating H3N2 influenza virus strains involving gene segments coding for the internal proteins occurs naturally in the human population and that this mechanism of genetic reassortment may be important in virus evolution and pathogenicity. In addition to antigenic drift of the HA protein, emergence of new epidemic H3N2 strains may be influenced by the establishment of a suitable RNA segment constellation through a combination of genetic mutation and reassortment between cocirculating viruses. This study establishes the importance of analyzing the entire genome of human influenza viruses when studying new epidemic strains. Changes in the internal proteins, as well as antigenic variability in the surface glycoproteins, should be considered when analyzing and predicting newly emerging influenza viruses in humans.