Differences in disease outcome between LPAIV and HPAIV infected chickens are obvious. Less obvious is the exact mechanism behind disease development after HPAIV infection. In humans, a cytokine storm (hypercytokinemia) with devastating consequences for the infected organs is thought to contribute to mortality after high pathogenic H5N1 infections [29, 30]. Indeed, the high mortality rate of H1N1 during the 1918 influenza pandemic was related to severe pathobiology of the lungs .
In accordance to work on H7N3  no major differences in cytokine mRNA levels in the lungs of chickens between H7N1 HPAIV and LPAIV infections could be found during the first 24 h of infection . In fact, only small pathological differences in lung and trachea were found between the two genetically related, but pathogenically distinct variants. Löndt et al.  demonstrated for Pekin ducks, that localization of H5N1 HPAIV in heart and brain tissue preceded death and the fact that these are vital organs correlated well with the finding of rapid mortality after HPAIV infections. So, assuming that differences between H7N1 HPAIV and LPAIV infections might be related to the differences in virus localization, we were surprised by the presence of H7N1 LPAIV RNA in organs beyond the respiratory and gastrointestinal tract, where the necessary proteases to cleavage LPAIV are absent. The observation that H7N1 LPAIV RNA could be detected for a prolonged time in multiple organs of all infected chickens indicated that the virus could spread systemically after an intranasal/intratracheal infection. The systemic spread of LPAIV could not be confirmed by immunohistochemistry (IHC) at 2 d.p.i. While all analyzed organs become positive for the virus nucleoprotein after an HPAIV inoculation virus could only be detected in the lung after an LPAIV infection (data not shown). The discrepancies between the qPCR and IHC could be due to differences in measurement, but are probably due to differences in sensitivity. The presence of H7N1 LP virus in brain was confirmed after inoculation of brain homogenate supernatant from LPAIV infected chickens in embryonated eggs and sequencing. Virus was demonstrated in the allantoic fluid by both HA-assay and qPCR. Furthermore, differentially regulated host gene expression in chickens infected with LPAIV was detected in both ileum and brain.
Thus in two separate experiments H7N1 LPAIV RNA could be found systemically. Although unexpected, our data were in line with findings for H7N1 LPAIV in turkeys by Toffan et al. . They found that besides lung, viral RNA was also detected in breast, thigh and blood. In addition, also H9N2 and H5N2 LPAIV were found in chicken organs that were not related to the respiratory and gastrointestinal tract [12, 13, 15]. Until recently, however, such observations were considered obscurities. The question arises how the presence of LPAIV (RNA) in organs that were expected to be infected by HPAIV only, are in proportion with the differences in mortality between the two strains.
Although brain and ileum are both infected, differences between these organs exist in the availability of the LPAIV cleavage proteases. Chicken brain lacks the required trypsin-like proteases . In line with the absence of these proteases in brain, the differences in viral RNA load between HPAIV and LPAIV infected chickens were considerable. The differences between HPAIV and LPAIV infected chickens were also visualized by the differences of the amount of affected genes in the brain. Similarly, the availability of proteases for both HPAIV and LPAIV cleavage in the ileum might be responsible for the limited difference in viral RNA load and the large overlap in regulated host genes between HPAIV and LPAIV infected chickens. Taking the data of brain and ileum together this could mean that differences in host responses and possible consequences in pathogenicity and mortality are merely determined by the amount of viral RNA or virus instead of other different characteristics between the strains (e.g. PB1 etc.). This hypothesis is supported by the observation that H7N1 HPAIV and LPAIV were closely related and that the differences between the two strains were predominately caused by the differences in the HA cleavage and nearby glycosylation sites .
Apart from the differences between high- and low pathogen virus infections, remarkable differences were seen between their effects on the brain and ileum. Remarkable, because differences between the organs were not only seen in the viral RNA load and amount of genes regulated, but also in the host response pathways that were affected. Despite the considerable viral RNA load in the brain of HPAI infected chickens, only two pathways were found to be significantly affected. For ileum at least six affected pathways could be found after analyses of the expression data. The Phosphatidylinositol signaling system, with was activated in both brain and ileum, is in mice important for signal transduction . The detected up regulation of genes of this pathway, might indicate increased cellular activity. The Toll-like receptor signaling pathway, which was regulated in the brain only, has a possible function in clearing the infection. Down regulation of genes of this pathway in the first 24 h, perhaps caused by elements of the influenza virus itself, might hamper the immunological response against the virus, thereby favoring the infection. In ileum, the pathways that are affected after an AIV infection are more diverse, but might be related to tissue regeneration. Possibly, AIV is a lesser threat to ileum because of the fast regeneration of the villus epithelial cells as suggested for Rotavirus by Snodgrass et al. .
Despite the differences in the amount of genes regulated, no remarkable discrepancies between LPAIV and HPAIV infected chickens were found for PER2, STC2 and FKBP5 by qPCR. PER2 is expressed in many brain areas and peripheral tissues of mammals and is generally associated with the circadian rhythm . PER2 is also linked to IFN-gamma regulation since PER2-deficient mice had an impaired IFN-gamma production . In the brain of LPAIV infected chickens PER2 was already down regulated at 8 h.p.i. Whether the down regulation of this gene forms the kick-off for other genes is currently unknown. Little is known about STC2, the paralog of the mammalian counterpart of the fish calcium-regulating hormone STC1, except for the fact that over expression might protect cells from apoptosis . STC1 is expressed in multiple organs modulating the immune/inflammatory response . FKBP5 is expressed in a variety of mouse and human tissues and has been shown to interact with various immune response pathways. In brain immunophilins like FKBP5, are modulators of the cortisol-HPA axis response to stress and related chronic brain disorders . In a ferret AIV infection model, it is suggested that the up-regulation of FKBP5 is a physiological response of lung cells to the increase of glucocorticoid, which facilitates the suppressive effect of glucocorticoid on pro-inflammatory cytokine production . Overall, an AIV infection in chicken brain resulted in regulation of genes that are frequently associated with immune regulatory functions, perhaps favoring infection or preventing tissue damage.
Virus is transported through the body via the blood stream [12, 43] and in our experiments PBMC were positive for viral RNA after an HPAIV or LPAIV infection (data not shown). Therefore, we cannot exclude the possibility that the viral RNA that we detected by qPCR resulted from the presence of blood in the organs examined. Furthermore, although viral induced gene expression might take place in the brain  the data on host gene regulation might also be derived from endothelial cells in the brain instead of from brain cells itself [45, 46]. Therefore, embryonic brain cell cultures were used to evaluate whether H7N1 LPAIV could infect cells derived from embryonic brains.
Brain cell cultures revealed that LPAIV, when grown in eggs, could infect embryonic brain cell cultures including astrocytes. LPAIV could also productively replicate in these brain cell cultures, as was seen by an increase in staining of viral nucleoprotein, after removal of the virus. However, progeny virus produced by these cells could only infect fresh brain cell cultures after treatment with trypsin. This indicates that cleavage by trypsin-like proteases was essential for the virus to become infectious. Since H7N1 LPAIV was grown in eggs, we speculate that it contains cleaved HA and is therefore able to infect secondary brain cell cultures and, to some extent, replicate in those cultures. In the absence of trypsin-like proteases however, the newly formed virus remains arrested in its native form, unable to infect fresh cultures. This hypothesis was supported by experiments with a prolonged culturing time: no additional staining, not to mention cytopathogenic effects, were found when the incubation was prolonged after the first 24 h of incubation (data not shown). By adding trypsin, the arrested virus is cleaved and infectivity is restored. In a similar way the isolated virus from homogenated brain samples might be detected. Since the necessary proteases are absent in the brain, the virus may in vivo also be present in an arrested form. Trypsin-like proteases in the allantoic of the embryo might be responsible for the (restored) reproducibility of the virus.
The in vitro work above may reflect the in vivo situation. Cleaved H7N1 LPAIV from primary infected organs like lung and trachea might infect many different organs via the blood. Massive replication in the organs with trypsin-like proteases, makes the virus easy to detect, while arrested virus, in organs with no or hardly any trypsin-like proteases, remain below the detection levels of relative insensitive assays. This hypothesis is supported by the results of a study in turkeys of Toffan et al. . Albeit not discussed, the sensitivity of the test method appeared to be very important in this study: only RT-PCR demonstrated H7N1 LPAIV RNA in each of the tested organs. Since more LPAIV strains were incidentally detected beyond the respiratory and gastrointestinal tract, we consider this feature not to be restricted to H7N1.
To date, all virulent influenza viruses tested have the ability to induce apoptosis in vitro . This led to the hypothesis that apoptosis may contribute to the lethality of the host due to viruses that can replicate in a variety of tissues. Indeed, specific apoptosis in the brain of HPAIV infected chickens correlated well with the fact that only HPAIV could replicate in the brain. Apoptosis was already seen 1 d.p.i., when the first chickens showed signs of illness (data not shown). Therefore infection of brain with HPAIV likely contributes to the rapid mortality after infection.