Influenza viruses are characterized by rapid mutation caused by error-prone viral RNA-polymerase enzyme on the negative-RNA genome during the replication. In addition, the segmented genome of influenza viruses allows these viruses to obtain novel genetic information by reassortment with other influenza strains. Error-prone RNA polymerase activity may be beneficial for the virus by providing diverse gene mutations that may allow rapid adaptation to a new host. Reassortment may also enable the virus to acquire features from other influenza viruses within short infection circles, thereby possibly rapidly adapted to new host population and becomes endemic or pandemic. Many influenza pandemics in history were caused by reassortant viruses originating from mammalian-adapted viruses that obtained genes from avian influenza viruses. For instance, the H1N1 virus causing the 1918 pandemic was a reassortant virus, containing HA from H1 subtype human-adapted strain and NA and other gene segments from avian influenza viruses [
55]. Also the viruses causing the 1957 pandemic [
56] and the 2009 pandemic [
57,
58] were reassortant viruses.
Considering the pandemic threat of avian influenza viruses, it is important to provide more insight in the characteristics of human influenzas viruses after reassortment with avian influenza viruses. To approach this, we simulated the reassortment of human H1N1 virus (inner genes) and avian H9N2 virus (both HA and NA genes, and one of the polymerase genes). The genotype of the H9N2 strain (A2093) used in this research was B69 with its HA gene clustered in Ck/BJ/1/94-like lineage [
59]. Besides, it has been reported with higher avidity for α 2,6- sialic acid. This virus strain showed ability to replicate in mammalian cells and in mice. [
60]. The surface genes were derived from the avian H9N2 virus, and the reassortant viruses thus were able to replicate but not adapted to the mammalian receptor as well as the wild-type human H1N1 strain. The reassortant viruses in this research were used as a model to study the early stage of evolution of avian-human reassortant influenza viruses. We showed that the RNP complex with the PB1 gene from A2093-H9N2 in the background of the WSN-H1N1 virus significantly promoted the activity of RNPs complex in a Dual-Luciferase Assay System. In MDCK cells, the PB1-reassortant virus was found to replicate with similar efficiency as wt WSN-H1N1 virus. In DF1 cells, the replication of the PB1-reassortant was reduced compared to wt WSN-H1N1 virus. The other reassortant viruses replicated less efficiently in both MDCK and DF1 cells compared to wt WSN-H1N1 virus. The reassortant viruses were able to replicate efficiently in the mouse turbinate and lung, to similar levels as wt WSN-H1N1 virus. Except for the PB2-reassortant virus that showed significantly reduced replication in the mouse lung. The body weight of the mice was measured as an indication for virulence (replication ability) of the viruses. Most interestingly, we observed increased virulence (replication ability) in mice for the PB1-reassortant virus. Due to the low pathogenicity of the virus, no mortality other than decrease in body weight were observed in the infected mice. A previous study detected a high polymerase activity of the combination of mammalian PB2 gene and avian PB1 gene in human cells [
61]. The involvement of avian PB1 gene in mammalian-adapted virus might obtain a higher virulence in new host by generating adaptive mutations under a new selection pressure. To obtain more information on the replication and evolution of the WSN-H1N1 virus containing inner gene segments of the avian H9N2 virus, we performed serial passaging of the reassortant viruses in MDCK cells and in mice.
In this study, the “mutation rate” was calculated during serial passaging of the reassortant viruses. The mutation rate is therefore a combination of initial errors made during RNA replication, combined with the effects of host selection [
62]. Mutations in HA may lead to changes of antibody or receptor binding, and may be preferentially selected [
63,
64]. For all reassortant viruses in this study, we observed a higher mutation rate (mutation/site/infection cycle) in mouse lungs compared to MDCK cells. This difference was likely caused by the increased selection pressure mediated by the mouse immune system. However, the absolute mutation rate measured for the viruses may differ dependent on the host species. The mutation rate measured on HA gene was more than two-fold increased for the PA-reassortant virus compared to wt WSN-H1N1 and A2093-H9N2 viruses, and more than 1.5 times for the PB1-reassortant virus. Previous studies suggested the mutation rate of influenza A viruses ranged from 7.1 × 10
−6 to 4.5 × 10
−5 substitutions per nucleotide per cell infection cycle (s/n/c) of the whole genome [
65,
66]. In this study, we measured mutation rates of 5.0 × 10
−4 and 6.2 × 10
−4 mutation/site/infection cycle on HA gene for the PB1 and PA-reassortant virus, which is higher than previously reported. We showed that polymerase activity was increased for the PB1-reassortant virus, which may have resulted in an increased error-rate during RNA replication. However, decreased polymerase activity was measured for the PA-reassortant. The higher mutation rate observed therefore may also result from the strong selection pressure on the reassortant viruses due to their novel genetic composition. We analyzed the HA sequence in this study, and higher selection pressure may be expected for virus surface protein [
67]. Furthermore, the mutation rate may not only depend on the gene segment analyzed, but also on the virus subtype as was reported previously [
67,
68]. Finally, differences in the analysis methods may have contributed to variation in the error-rates reported [
69,
70]. The number/ratio of non-synonymous mutations is indicator for the selection pressure on the virus [
71]. With similar high SBS numbers, the PB1-reassortant virus showed the lowest precentages of non-synonymous changes, whereas in PA-reassortant virus only non-synonymous changes were found. This high percentage of non-synonymous mutations in PA-reassortant virus was also reflected in a high relative genetic distance, suggesting there is a strong positive selection on the PA-reassortant virus. The serial passaging experiments, in which five host-infection circles were observed, showed that the substitution rates of both the PA and PB1-reassortant viruses were increased compared to the other reassortant viruses. This suggests that reassortant virusses obtaining the PB1 gene from avian H9N2 are more likely to rapidly adapt to new hosts. This in accordance with a previous study which showed that virus replication was more efficient when PB1 was derived from an avian virus, regardless of the origin of the other proteins [
72]. Furthermore, we identified a mutation hot-spot in the HA-gene, that is located near the antigenic and receptor binding sites [
73‐
75]. We measured a significantly increased substitution frequency for the 350–950 domain of HA1 compared to the complete ORF region. Our results are consistent with previous studies which showed that the head domain of HA evolves faster than the stalk domain [
76]. This domain included the 130 helix and 220 loop structure of HA head which are exposed to the surface and therefore can be easily captured by host immune system [
77]. Together with the receptor-binding function, the highly mutable HA1 domain might compromise viral replicative fitness, which means the globular head of HA are highly tolerant of mutations [
78]. We further indicated that the mutation patterns could be highly influenced by the reassortant viral vRdRp complex, especially in reassortment between human and avian viruses. However, further research will be required to provide more insight in the intracellular mechanism at molecular level.
Reassortment events between human and avian influenza viruses in combination with rapid evolution and adaptation due to error-prone replication may lead to a novel human pandemic. The H9N2 virus is currently the most frequently detected subtype (particularly in live bird markets) and has become endemic in poultry across Asia since 1990s [
79]. Several studies provided evidence of interspecies transmission of H9N2 virus from poultry to mammals, such as swine [
80,
81]. Swine may represent “mixing vessel” for influenza viruses as they are susceptible for infected with swine, human and avian influenza viruses [
82]. An experimental study showed the replication of H9N2 virus (A/guinea fowl/Hong Kong/WF10/99, A/guinea fowl/Hong Kong/NT184/03) in mice without adaptation [
83], likely because of its properties of internal genes related to polymerase function. As human infections with avian H9N2 viruses have been reported [
8,
14], there is a high probability of reassortment with human influenza viruses.