The current study was designed to assess the effect of genetic variation in the host on survival after infection with a highly pathogenic H5N1 influenza virus. Applying the murine model of H5N1 influenza virus infection, we showed that genetic variation significantly contributes to survival. QTL mapping identified five loci associated with severity of disease after H5N1 virus infection, indicating that multiple genes are responsible for the observed difference. RNA expression analysis has resulted in several candidate genes whose function is potentially linked to infection or inflammation.
Following the initial observation that certain mouse strains are more susceptible to severe disease after infection with highly pathogenic avian H5N1 influenza A virus but that other strains are resistant to disease, we sought to identify the underlying mechanism responsible for this difference. Susceptible mice that succumbed after infection had high viral loads and increased production of proinflammatory cytokines early after infection. In contrast, resistant mice that effectively cleared the infection had reduced virus titers and produced lower levels of proinflammatory cytokines. This link between disease severity and high viral loads plus increased production of proinflammatory cytokines was previously found in humans infected with H5N1 viruses (12
). de Jong et al. (12
) observed in a cohort of H5N1 virus-infected individuals that high viral loads in throat swabs and the presence of high concentrations of TNF-α and CCL2 in serum were associated with poor prognosis. In studies comparing pathogenicities of different influenza A virus strains, high viral load combined with increased levels of proinflammatory cytokines was also associated with severe disease and increased mortality (22
An important and novel finding of this study is that H5N1-induced pathology is greatly affected by genetic polymorphisms in the genome of the infected host. We have also found that, at least in mice, H5N1 pathogenesis is a complex genetic trait with multiple genes affecting disease outcome. This result is reminiscent of many other microbial diseases including human immunodeficiency virus infection and Mycobacterium tuberculosis
). A possible explanation for the observed complexity is the nature of the pathogen, e.g., viral, requiring a host cell for propagation. Following attachment, fusion, replication, and assembly of progeny virus in the host cell, an innate and adaptive immune response is initiated to eliminate the pathogen in a time- and energy-efficient manner. The many genes involved in this process can all greatly influence the outcome of the disease. Our approach of combining QTL mapping with RNA expression analysis at day 3 has shifted the focus to identifying genes involved in viral replication or early innate immune responses. However, the identification of Hc
, the candidate gene on Qivr2
, is an example of a protein involved and required for effective adaptive immune responses.
The highly pathogenic A/Hong Kong/213/03 influenza A virus was used to identify host genes and gene networks promoting survival after infection. It was selected from several H5N1 viruses for its extremely large difference in LD50
(>10,000-fold) between the two parental mouse strains B6 and D2. Other H5N1 viruses, including A/Vietnam/1203/04 virus, demonstrated increased lethality to B6 mice, effectively reducing our difference in LD50
and potentially compromising our genetic screen. Preliminary studies were also done with the mouse-adapted A/Puerto Rico/8/34 virus (PR8) (data not shown). For this particular virus, the difference in LD50
between D2 and B6 mice is approximately 100-fold (data not shown) (32
). Also, we measured survival in 13 BXD RI strains after infection with an intermediate dose of PR8. BXD strains with severe PR8-induced pathology were also among the most susceptible to HK213 virus, suggesting that some of the candidate genes identified in this study are important for survival after influenza virus infection in general. The identification of Hc
supports this, as it was first shown to be important for survival after infection with A/PR/8/34 influenza A virus.
It is tempting to speculate that certain genetic deficiencies identified in this study are universally associated with increased susceptibility to influenza virus and potentially other viruses like Sendai virus. An example of this is Hc
, which was shown to affect survival after inoculation with a highly pathogenic H5N1 influenza virus, supporting a previous publication demonstrating a similar effect of Hc
after inoculation with PR8 or X31 (an H3N2 virus with the internal genes of PR8). Our study, however, is also likely to identify genes whose deficiency affects specifically H5N1 influenza A virus-induced disease because of the difference in pathology between highly pathogenic and low-pathogenic influenza A viruses (4
To identify candidate genes within each of the five Qivr
, we analyzed the pattern of RNA expression. This methodology has been effective in the past and allows for the identification of genes responsible for infectious diseases or immune system-related phenotypes (16
). One of the three candidate genes on Qivr2
, was previously shown to be involved in both the innate and adaptive immune responses to infectious agents including low-pathogenic mouse-adapted influenza viruses (20
). We have now shown that Hc
is also important for adequate adaptive immune responses to H5N1 viruses, as indicated by the higher viral loads in Hc
-deficient mice (Fig. and ). Although this is not surprising, considering the previously identified role for Hc
in the inflammatory response to low-pathogenic viruses, the confirmation illustrates the usefulness of RNA expression analysis to identify the gene within a QTL responsible for the increased resistance to infection.
The identity of the underlying genetic variation in the other three loci (Qivr7
, and -17
) is currently unknown; however, we have identified several candidate genes based on expression levels, the presence of a single nucleotide polymorphism encoding an amino acid change, and previously published literature. The main candidates for Qivr7
), and Gvin1
(very large interferon-inducible GTPase). Trim30
limits the response of macrophages to Toll-like receptor stimuli through binding to TBK1 (40
); other Trim proteins were shown to have antiviral properties (10
). Several Trim genes, including Trim30
, were upregulated in macrophages and dendritic cells maintained in culture upon exposure to influenza A virus (H1N1) (37
). For Qivr11
, we identified three potential candidate genes: Grn
, and Dhx58. Ifi35
is induced upon exposure to interferon and is reported to modulate cytokine signaling (49
also has antiviral properties against bovine foamy virus (41
contains several candidate genes including Eif2ak2
, two genes with known roles in influenza virus infection (1
). However, neither protein contains any single nucleotide polymorphism encoding an amino acid change. Other candidate genes on Qivr17
(elastin microfibril interfacer 2), encoding a protein that has affinity for the protective antigen of the anthrax bacterium and is involved in the extrinsic apoptosis pathway (14
), and Ipaf
, an intracellular pattern-recognition receptor (18
A more detailed analysis of expression levels and identification of important gene networks differentially expressed following infection enable further selection of genes among the relatively large number of candidate genes. A significant drawback of this approach is that the gene of interest must demonstrate a difference in expression level, certainly not a prerequisite for differential activity, and that the gene of interest or a splice variant must be detected by the microchip. When genetic changes affect function but not expression level, downstream effects of this particular change can then be used to identify the gene of interest. Overall, this technique, despite its limitations, is considered an important tool for selecting candidate genes within a QTL and one which we have used here.
Collectively, our results show that host genetic variation has a significant impact on survival after infection with a highly pathogenic H5N1 influenza A virus, which is associated with lower viral loads and reduced levels of proinflammatory cytokines, both of which are hallmarks of a mild H5N1 virus infection in humans. Gene mapping and RNA expression analysis have identified several candidate genes, whose genetic variation may ultimately determine the outcome after infection. Our study has convincingly shown a dramatic effect of host genetics on H5N1 virus infection, supporting those studies claiming a genetic predisposition in humans. This study provides a list of priority genes which should be closely examined in family clusters of H5N1 virus infection in humans.