The rIndo and rEgret viruses, although having a high sequence similarity (>91%) for all eight gene segments, had a marked difference in virulence levels in chickens. Using reverse genetics, a series of variant viruses differing by a single gene segment was created in order to explore the contribution of individual viral genes to viral pathogenesis in chickens. Unexpectedly, the reassortment of the NP gene resulted in the biggest differences in virulence and replication. Some increase in pathogenicity was seen with the exchange of the HA, NS, and M gene segments, while the PB1 and PB2 reassortants had impaired replication resulting in low virulence. Alterations in host gene expression in response to infection with the reassortant viruses suggest that the viruses use different mechanisms to evade host responses.
HA is known to be an important determinant in the virulence of AIV (9
). The HA genes of both H5N1 recombinant parent viruses contain the same multiple basic amino acid motif adjacent to the cleavage site. However, there were 11 other amino acid differences between viruses outside of the cleavage site of the HA gene, 5 of them localized in the receptor binding domain. The increased pathogenicity of the rIndo/rEgret HA virus over the rIndo parent may therefore be attributed to mutations outside of the multiple basic cleavage site, possibly in the receptor binding site.
Since we were not able to rescue the rIndo/rEgret NA reassortant, the role of NA could not be explored in this study. One possible reason may be that HA-NA incompatibility is responsible for inefficient replication of the rIndo/rEgret NA reassortant virus in 293T cells or ECEs. The HA and NA proteins of influenza work together to efficiently bind and release virus from the cells during replication, and it is known that HA-NA incompatibility can result in inefficient viral replication or aggregation on the cell (10
). The importance of a balanced HA-NA relationship often results in the coevolution of NA along with changes in HA of influenza viruses to maintain a functional unit (34
). There is some evidence the common chicken-adapted NA stalk deletion may result in steric hindrance causing inefficient virus release (34
). The rIndo chicken AIV has a 20-amino-acid NA stalk deletion, while rEgret virus NA does not carry this deletion. The importance of possible HA-NA incompatibility warrants further study.
Previous studies have shown that the M gene from A/Mallard/78 attenuates the H3N2 human viruses in squirrel monkeys and humans (13
). M has not previously been identified as an important virulence factor in pathogenesis in chickens. The matrix protein gene consists of spliced mRNA products encoding M1, an internal structural protein, and M2, an integral membrane ion-channel protein (58
). Our recombinant viruses have identical M1 protein sequences allowing us to further attribute that the increased pathogenicity was due to the three amino acid changes in the M2 sequence. The M2 ion-channel protein is thought to stabilize the HA proteins, especially H5 and H7 HA proteins with multiple basic cleavage sites, by maintaining the pH so that the conformation of HA into the low-pH form does not occur prematurely (12
). The mechanism by which rEgret M2 increases the pathogenicity of the rIndo virus may be the result of the increased stabilization of rIndo HA so that attachment and replication of the rIndo/rEgret M virus are more efficient, therefore allowing the spread to a larger range of tissues than was seen with the viral antigen staining (Table ). However, viral replication data from lung and spleen tissues do not appear to support this explanation, as we did not see higher titers of rIndo/rEgret M than of rIndo in those tissues in infected chickens (Fig. ). However, titers were measured at only one time point and may not reflect titers throughout infection.
The effect of the M2 protein on virulence has been widely studied, as it relates to resistance to the antiviral drug amantadine, which blocks M2-ion channel activity (23
). One of these studies reported that the S31N mutation increased the virulence of the A/WSN/33 virus in mice as well as conferred amantadine resistance (1
), and another study reported that the S31N mutation reduced the activity of A/Chicken/Germany/34 M2 (20
). Based on these previous studies, we expected that the rIndo virus containing an N at position 31 of M2 would result in a more virulent phenotype for chickens; however, that is not the case. When rEgret M2 with the reportedly less virulent S at position 31 replaced rIndo M2, the rIndo/rEgret M virus showed increased virulence and tissue distribution. It is possible that one of the other two amino acid changes in M2 has a greater contribution to virulence or that the other mutations suppress the S31N mutation in rIndo M2, resulting in a less virulent strain. The mechanism by which M2 increases the virulence of rIndo/rEgret M needs further exploration.
Influenza NP is important in the packaging of the viral RNA and has been shown to be involved in many aspects of viral replication (41
). Small interfering RNA against NP resulted in decreased viral replication in ECEs and in mice (18
). It has previously been demonstrated that NP directly interacts with viral PB2, PB1, other viral NPs, and also other cellular factors (7
). Previous studies have shown viral replication to be more efficient when the NP and polymerase genes are derived from the same virus, indicating viral replication requires the proper combination of genes to function properly (37
). The NP gene has also been shown to be important for host range restriction for A/Mallard/78 or A/Pintail/Alberta/119/79 and has also been shown to attenuate the H3N2 human viruses in squirrel monkeys (13
). NP has not previously been identified as a sole virulence or replication factor in the pathogenesis of AI in chickens. We show here that the exchange of NP was sufficient to greatly increase replication, tissue tropism, and virulence and alter the expression of selected host genes in chickens. The mechanism by which NP increases virulence remains unclear. Our reassortant viruses have five differences in the NP amino acid sequence. One mutation, at position 22, spans both the RNA binding and PB2-1 binding domains of NP (41
). Three of the other mutations (positions 400, 406, and 423) are located in the overlapping regions of the NP-2 and PB2-3 binding domains, suggesting that it is either the NP or PB2 binding function that is resulting in the increased replication (41
). One possibility is that the altered NP-PB2 interaction of the rIndo/rEgret NP virus results in more efficient binding in the polymerase complex that aids increased replication, allowing the virus to spread more efficiently to all tissues, resulting in a more severe systemic disease.
PB1 has been shown to be associated with the high pathogenicity of some H5N1 viruses in ducks (26
), and the alternate splice product, PB1-F2, has been shown to be important in the virulence of human fatal case viruses in mice (14
). PB2 has been shown to contribute to the virulence of AIV, with a lysine residue at amino acid 627 being linked to the increased virulence of H5N1 viruses (22
). Neither of our recombinant viruses has the lysine at amino acid 627 in PB2, suggesting that this residue is not important for pathogenesis in chickens. The polymerase complex (PA, PB2, and PB1) has been shown to work inefficiently when the components are derived from different host viruses (13
), indicating that this relationship is vital to replication and that these proteins may adapt together to maintain functionality (39
). While both of the recombinant viruses that we used were avian in origin, some incompatibility may occur when the polymerase complex components are derived from two different parent viruses. Further studies will help determine the role of compatibility among the polymerase genes in viral replication.
In accordance with previous studies, we found that PB1 and PB2 are important for efficient virus replication (Table ; Fig. ). PB1 interacts with PB2, PA, and NP, and the multiple binding interactions may explain the lower titers of the rIndo/rEgret PB1 virus. The PB1 gene of more than one human pandemic virus is known to be derived from avian viruses (29
). None of the five amino acid differences are in known binding domains of PB1. For PB2, analysis of the binding domains of PB2 indicates that the NP, PB1, and cap-binding (24
) functions could be affected by the 105, 132, 221, 251, 389, or 394 amino acid changes that fall in those binding domains. Disrupting the functional polymerase complex unit is not favored for optimal replication efficiencies, as seen by the decreased distribution, decreased tissue staining, and decreased viral titers in tissues (Table ; Fig. ).
Regulation of host gene expression may in part explain the mechanism used by some of the reassortant viruses to evade the host immune response. IFN-α, a cytokine previously shown to have an antiviral effect on influenza viruses (33
), was downregulated in both lung and spleen tissues infected by rIndo/rEgret HA and rIndo/rEgret NS compared to the level in lung and spleen tissues infected by the rIndo parent (Fig. ). Downregulation of antiviral IFN-α may be one of the mechanisms that these viruses use to evade the host responses and may contribute to the increased replication of the viruses in tissues, leading to the increased viral antigen staining that we saw (Table ). Infection with rIndo/rEgret PB1 and rIndo/rEgret PB2 viruses resulted in upregulation of IFN-α in lung tissue (Fig. ). The increased IFN-α levels may explain why these reassortants do not show increased virulence and have decreased replication in tissues (Table and Fig. ). The upregulation of IFN-α in the lungs may have been enough to fight infection at the site and prevent the systemic spread of the virus. The D92E NS1 mutation has been shown to increase resistance to IFN in pigs (48
); however, none of our recombinant viruses harbors this mutation. The fact that the replication of the rEgret and rIndo viruses in the spleen was not affected by IFN-α upregulation suggests it is one of many factors that the host can regulate to inhibit influenza infection.
Upregulation of the Mx
gene has been shown to increase with IFN-α expression, and there is some evidence that this may help combat influenza in mammals (17
). In a chicken cell line, chicken Mx1
did not result in antiviral effects against influenza (6
); however, it was later found that Mx1
is a polymorphic gene and that the Mx protein from different breeds of chickens can in fact have antiviral properties against influenza (30
). Our results indicate that Mx1
is strongly downregulated in the lungs and spleens of rIndo/rEgret PB1- and rIndo/rEgret PB2-infected chickens (Fig. and ), yet there is no replicative advantage in downregulating Mx1
for these viruses (Fig. ; Table ). Therefore, Mx1
gene expression levels in the lung and spleen did not appear to correlate with the virulence of our recombinants in chickens and support previous findings that chicken Mx may not have antiviral activity in all chicken breeds (30
IFN-γ has been shown to increase the secretion of reactive oxygen species such as nitric oxide (NO) (2
), and there is some evidence that NO has antiviral properties (3
). In the spleen, rEgret and rIndo/rEgret NP infection resulted in an increase in iNOS and IFN-γ gene expression. However, only the rIndo/rEgret NP virus showed substantial viral replication in the spleen. The increased NO production may reflect the rapid replication of the rIndo/rEgret NP virus and the host's attempt to prevent the replication. The short MDTs and increased tissue lesions resulting from infection with these viruses may be due to the NO (Table ; Fig. ). In particular, rIndo/rEgret NP caused extremely short MDTs and massive replication of the virus in tissues (Table and Fig. ). The rIndo/rEgret NP virus not only increased IFN-γ and iNOS expression in the spleen but was the only virus studied that upregulated these genes in the lungs as well, which also may explain in part the increased virulence of this strain in chickens.
In summary, the pathogenicity of AIVs is clearly due to a polygenic effect, and in this study, single-gene changes led to restriction or increased replication of AIVs in chickens. Reassortment events can result in a number of different gene combinations; however, we, and others, have shown that all possible combinations of genes in reassortant viruses are not necessarily viable. From the remaining viable gene reassortants, we were able to determine the effects of single-gene changes on the replication and pathogenesis of AIVs in chickens. While the precise role of chicken immune response factors is not clear, changes in the host gene expression of several genes involved in immunity are influenced by infection with the reassortant viruses and may be contributing factors in the pathogenicity of AIVs in chickens. Based on the results obtained in this study, the next step is to investigate the role of individual amino acids in each of the genes shown to affect AIV pathogenicity, as well as to continue exploring the effect of certain gene combinations.