Here we present the heterogeneous virulence of three different strains of influenza H1N1 in human adenocarcinoma cells (A549) and mice that were isolated from clinically similar human cases from South China in December 2009. Two different patterns of biological heterogeneity were observed: first, two strains (NC2 and NC11) showed efficient viral replication and subsequent effects on tissue histology, induction of proinflammatory response and causing lethality in mice, although their behavior was not totally identical and some minor differences in the kinetics of the disease in mice were observed. Secondly, NC8 showed delayed replication that eventually led to non-lethal infection and muted inflammatory response in mice. These results have a relevance to the previously published epidemiological reports that associate effective viral replication and delayed clearance with disease severity in humans [9, 15, 16]. Most of the previous studies agree that pdm H1N1 exert homogeneous and modest infection but with efficient pulmonary viral replication in mice ; however, its pathogenesis is more than that of seasonal strains [5, 18] and subdued compared with 1918 pandemic and other swine origin influenza viruses [17, 19]. Nonetheless, the virus has been shown to increase virulence upon expression of truncated viral proteins by reverse genetic tools and after mice adaptation . In addition to the heterogeneous nature of these strains, we also demonstrate that C57/BL6 mice are more susceptible for pdm H1N1 infection than BALB/C strains; however, variation in disease kinetics did not change the infectivity ratios as observed previously .
In this study, NC2 and NC11 viruses were able to induce of proinflammatory cytokines effectively whereas immune responses were mute in the case of NC8 infection. It is interesting to note that pdm H1N1 strains also display a differential cytokine response which may or may not be linked with viral growth. Previous studies have shown a robust gene expression of innate immune response genes with delayed switch to adaptive immunity after pdm H1N1 infection; however, overall responses are considered to be higher compared with those of contemporary seasonal strains [7, 17].
Clinically, 2009 influenza pandemic caused self recovering mild disease in the vast majority of patients while only a small group of patients developed serious respiratory complications [21, 22]. No explanation has yet been offered for why the clinical profile varies from one patient to another. The published studies interrogating host markers and viral pathogenesis in vitro and in vivo are mostly limited to the characterization of a narrow range of prototypic pdm H1N1 strains [5, 17–19]. Consequently, important aspects of the disease may remain unexplored. Pandemics provide a greater chance for influenza viruses to mutate; however, unveiling their impact on viral pathogenecity is an enduring goal that can be achieved by continuous surveillance. Lab investigation of newer strains might provide valuable information about the pathogenesis that could be missing in initial studies.
In the present study, although all these viral strains were isolated from patients who finally recovered, the viruses were able to produce biological heterogeneity in mice that refute the common paradigm of the evaluation of influenza pathogenesis which is at present based on the clinical profile and disease outcome of patients. Such attributes have previously been observed in clinically relevant influenza H5N1 strains [23, 24]. In humans, viral heterogeneity may have specific effects on individuals with different genetic background and demography; therefore, infection with such viruses might result in a variable clinical course of infection. However, it is also important to remember that treatment strategies, immunocompetance, and clinical management influence the disease severity and outcome and consequently mask the true picture of viral pathogenesis.
Virulence and interspecies transmission of influenza virus is often considered a polygenic phenomena [25–28]. The triple reassortant pandemic 2009 influenza virus stands out from ancestral pandemic and reassorted strains because it rapidly transmits to humans despite the absence of any traditional virulence markers; for example, the C-terminal PDZ ligand domain of NS1 , functional PB1-F2 protein, and PB2- K627 . Therefore, efforts have been made to determine other possible virulence determinant(s). Recent laboratory investigations conducted with mouse adapted pdm H1N1 strains speculate the role of HA (D131E and S186P)  and PB2 genes, such as glutamate-to-glycine substitution at 158 , aspartate to asparagines at 701 , Threonine-to-alanine at 271  and second site suppressor mutation  in viral replication and mouse adaptation, although none of them was demonstrated in wild type strains. In this study, the genetic characterization showed that the non-lethal NC8 strain contained three mutations (PB2-V227I, R299K and PA-E243G) in polymerase subunits compared with virulent strains. Previous studies have reported that both PB2 and PA genes are genetically linked with each other ; furthermore, N-terminal mutations in these genes might lead to intermediate or complete loss of viral RNA transcription . Therefore, we might speculate that these mutations are interlinked and collectively responsible for altered replication of the NC8 strain. On the other hand, this virus (NC8) strikingly replicated more than other viruses with > log10 ratio in embryonated chicken eggs, indicating the ease of growth in an avian environment. Here it is important to consider that the 2009 pandemic virus contains polymerase subunits PB2 and PA of North American avian lineage. We do not know whether these substitutions in NC8 are the remains of ancestral avian strains or not, but upon sequence analysis of global pdm H1N1 isolates, we found that these amino acid residues (PB2-I227, K299, PA-G243) are conserved in pdm H1N1 strains, thus raising the possibility that collectively they have some role in the adaptation to the mammalian host and they might link to the heterogeneity of pdm H1N1. However, in vivo studies with mutant strains are required to prove the hypothesis. In the case of HA gene, NC2 and NC11 contained the A409V mutation compared to NC8 and prototype California strains. It is worthwhile to indicate that NC2 also exhibited HA-E391K, which has recently been identified as a fast-growing mutation with the ability to destabilize the HA oligomerization process, thus modifying the membrane fusion properties of the pandemic influenza virus [36, 37]. However, no association with virulence and progression of disease has been established yet. Taken together, we hypothesize that these mutations in PB2, PA and HA genes might have no relevance with human disease but in the case of zoonotic transmission of influenza viruses to human, it may yield more pathogenic viruses.