Viral adaptation is considered to be one of the major mechanisms for generating pandemic influenza viruses 
. However, the molecular basis of adaptation of influenza A virus to a new host is poorly understood. The present results showed that adapted virus could be obtained by serial passages and PB2 gene of SD16-MA is critical for the virulence of mouse-adapted H9N2 influenza virus, especially, the combination of PB2 M147L and E627K contribute to the highly lethality of mouse-adapted H9N2 influenza virus.
In the study, viruses containing SD16-MA PB2, HA or M gene all grew better in lungs of mice than SD16, whereas mutations in PB2 and M1 protein demonstrated increased viral replication ability in vitro
, but only the mouse-adapted PB2 gene significantly increased viral virulence. Thus all of the 3 mutant genes were demonstrated to possess increased replicative functions in mice indicating the functional basis for their selection on mouse adaptation. The PB2 gene has multiple functions, such as binding host capped mRNAs and initiating viral mRNA synthesis, making it an important pathogenic determinant 
. The amino acid at position 627 of the PB2 protein has been described as a host range determinant, and the mutation E627K has been shown to be a key factor in the adaptation of H5N1 
or other avian subtypes 
influenza viruses to mammals. Hossain et al
adapted a wildtype duck H9N2 virus in quail and chickens through serial lung passages. The adapted viruses were readily infect mice and resulted in a quick selection of PB2 E627K mutation 
. It has also been demonstrated that viruses with lysine at this position were lethal in mice, whereas those with glutamic acid were avirulent 
. However, in our study, PB2 E627K mutation did not significantly increase pathogenicity though it could increase viral replication in lungs of mice, indicating that the viral genomic backbone affects the phenotype of mutations at position 627. Herfst et al also found that introduction of E627K mutation to H1N1/2009 virus had no major impact on virus replication in the respiratory tracts of mice and ferrets or on pathogenesis 
. Another report showed that PB2 E158G could increase the morbidity and mortality of the H1N1 pandemic virus, which was much stronger than the effect of PB2 E627K 
. All these data suggested that the virulence function of PB2 E627K seemed to be strain-specific or needed to interact with residue at other positions.
Most of the previous studies found major contributions of single amino acid mutations to viral adaptation and virulence, such as PB2 E158G 
, I504V 
, E627K 
, D710N 
, here, we demonstrated the importance of synergism of two or more amino acid substitutions in combination with E627K. Liu et al also found that a combination of PB2 271A with 590/591 SR polymorphism was critical for H1N1/2009 and triple reassortant swine influenza viruses for efficient replication and adaptation in mammals 
. In the present study, introducing single mutation of M147L, V250G or E627K to SD16 PB2 protein did not significantly increase the pathogenicity of virus in mice, nevertheless, substitution of two amino acid residues could increase the virulence, among which the 147L/627K combination caused significant mortality. Both PB2 147 and 627 positions were located in PB2 and NP interaction regions, where mutations at these two positions might complement each other and mediate enhanced protein function, leading to increased replication and mortality. However, this explanation needs to be further investigated by crystal structural determination of PB2-NP and possibly host protein complexes. The structure of C-terminal domain of influenza virus polymerase PB2 subunit has been solved to show that K627 is located on a large solvent-exposed face of the protein with a predominant positively charged surface 
. Nevertheless, the structure of N-terminal domain of PB2 protein is not known. So, the exact relationship of amino acids between position 147 and 627 in PB2 protein awaits further investigation.
Increased polymerase activity was considered to play a role in the adaptation of influenza viruses, because it corresponded to the optimal growth in mammalian cells, replicative fitness and virulence in mice 
. However, some mutations in the polymerase genes acted to enhance the polymerase activity, but not pathogenicity of virus 
. Song et al
reported that PA N383D contributed to the difference in the polymerase activities of two H5N1 viruses, but could not increase viral virulence, which may be affected by high level of polymerase accumulation in the nucleus of influenza virus-infected cells. Here, PB2 E627K increased the polymerase activity and replication of virus, but did not significantly increase morbidity, as did the combination of V250G and E627K. Combining mutations at 147 and 627 in PB2 could confer increasing polymerase activity and virulence to the adapted virus. These results suggested that high polymerase activity was a prerequisite for the high virulence.
Viral replication is thought to be an important and characteristic prerequisite for virulence. Some highly pathogenic H5N1 avian influenza viruses and wild-type H9N2 viruses that were lethal to mice without adaptation all replicated well in lungs, and even replicated systemically 
. The SD16 virus, used in this study, was avirulent to mice and only could be detected at lower titers in the lung of infected mice. After adaptation, SD16-MA virus gained higher virulence and replicative fitness in mice with major roles shown for M147L/E627K mutations that possessed higher virulence than SD16, accompanied by greater virus titers in lungs of mice. The SD16-M147L, SD16-E627K, SD16-V250G/E627K mutants also replicated efficiently in vivo
and in vitro
, but were not lethal for mice. These results suggested that high virus load was a prerequisite for the high virulence of virus in mice, but not sufficient. Other factors, such as polymerase activity and regulation of the proinflammatory response, also contribute to virulence of influenza viruses 
. Here, we observed that levels of IL-10 and IFN-γ in lungs of SD16-MA infected mice were higher than those induced by SD16 virus. The elevated levels of IL-10 and IFN-γ were also found in the peripheral blood of patients infected with H5N1 influenza virus 
. IL-10 plays an important role in the dampening of inflammatory response to prevent excessive damage to the host 
. IFN-γ is known to mediate the increased production of nitric oxide 
, which can subsequently result in the recruitment of more neutrophils and macrophages 
In summary, using a highly lethal mouse-adapted H9N2 virus, we demonstrated mutations in PB2 protein were critical for the adaptation and virulence of H9N2 virus in mice. Although the E627K mutation on its own enhanced replication and polymerase activity, it did not significantly increase pathogenicity of virus in mice. The combination of M147L and E627K in PB2 was critical for the high virulence of mouse-adapted H9N2 virus.