VN/1203 is more pathogenic than the 1918 virus in mice.
Our previous studies of macaques infected with either VN/1203 or the 1918 virus indicated that the 1918 virus is more pathogenic in this animal model (8
). However, of the 498 confirmed human cases of avian H5N1 influenza reported to the World Health Organization as of May 2010, 294 had resulted in death, representing a case fatality rate approaching 60%. Although there may be mild infections that go undiagnosed, H5N1 viruses appear to be significantly more pathogenic than the 1918 virus in humans (28
). Although the genetic, anatomical, and physiological similarities between macaques and humans suggest that macaques provide a highly relevant animal model, the mouse infection model is widely used because it provides an approximation of human disease; larger numbers of animals can be used to improve statistical significance; and, importantly, a wide variety of gene knockout animals are available. Therefore, to gain additional insights into the host response to these viruses, we used a mouse infection model that included wild-type mice and mice lacking the type I IFN receptor, a critical component of the innate immune response. When inoculated into wild-type 129S6/SvEv mice, both VN/1203 and the 1918 virus caused lethal infections; however, the mean time of death was 6 days for VN/1203-infected animals and 9 days for mice infected with the 1918 virus (Fig. ). Animals were sacrificed according to weight loss euthanasia criteria (see Fig. S1 in the supplemental material). Moreover, VN/1203 disseminated to extrapulmonary organs, as demonstrated by detection of virus in brain and spleen (Fig. ), whereas the 1918 virus was not detected outside the respiratory tract. The observed differences in viral dissemination and time to death could not be attributed to differences in viral titers in the lungs, as the two viruses were present at similar levels at all time points examined (Fig. ).
FIG. 1. VN/1203 infection is more pathogenic than 1918 infection in a mouse model of infection. (A) Mortality data from a total of 12 mice (6 mice/virus). (B) Viral dissemination data from a total of 12 mice (3 mice/virus/time point). d, day(s). (C) Lung virus (more ...)
Histopathology results indicated that two of six animals infected with the 1918 virus had mild epithelial necrosis in bronchioles and minimal peribronchiolar alveolitis at day 1 p.i. By day 3 p.i., three of the animals still lacked lesions and three presented mild to severe necrotizing bronchiolitis with associated neutrophilic casts in large bronchioles, peribronchiolar edema and inflammation, and moderate peribronchiolar neutrophilic-to-histiocytic alveolitis. By 4 days p.i., pneumonia and bronchiolitis were consistent in all animals and were slightly more severe than those observed at day 3 p.i. No lesions were observed in heart, spleen, brain, kidney, or liver in 1918-infected animals (see Fig. S2A and B in the supplemental material). Animals infected with VN/1203 presented at day 1 p.i. with moderate necrotizing bronchiolitis with neutrophilic inflammation and minimal peribronchiolar alveolitis. By days 3 and 4 p.i., the airway lesions were accompanied by minimal to mild peribronchiolar alveolitis. VN/1203-infected mice presented no lesions in extrapulmonary organs (see Fig. S2C and D in the supplemental material), but virus was detected in brain and spleen. Viral antigen was commonly present in bronchiolar epithelial cells and less commonly in alveolar epithelial cells and macrophages (see Fig. S2E and F in the supplemental material). Staining was more intense and more widely distributed in VN/1203-infected mice than in 1918 virus-infected mice. In summary, both viruses caused lethal infections, with death occurring approximately 2 days sooner among VN/1203-infected mice. VN/1203 disseminated to brain and spleen by 5 days p.i., but without noticeable tissue damage to those organs. The absence of pathology in extrapulmonary organs may be due to detection of recent virus replication that had not yet resulted in observable lesions. Alternatively, lesions may have been random and focal and not present on the samples examined.
VN/1203 elicits an earlier and more robust host transcriptional response.
In order to discover aspects of the host response that could explain differences in pathogenicity and dissemination, we used a functional-genomics approach to investigate the host transcriptional response elicited in the lungs of mice infected with VN/1203 or the 1918 virus. When analyzing the global transcriptional response, we found that VN/1203 infection induced the differential expression of numerous genes at all time points analyzed. Whereas the day 1 transcriptional response was attenuated in animals infected with the 1918 virus relative to the response induced by VN/1203, at later time points, the host responses to the two viruses were more similar (Fig. ). This more robust transcriptional response to VN/1203 correlates with our observations that all VN/1203-infected animals showed moderate airway lesions at day 1 p.i., although we have not directly evaluated the extent to which specific gene expression changes are responsible for lesion development.
FIG. 2. Global transcriptional responses to highly pathogenic 1918 and VN/1203 viruses. Shown is microarray analysis of whole lung tissue from 1918 and VN/1203 virus-infected wild-type mice at 1, 3, and 4 days p.i. The heat map illustrates the global transcriptional (more ...) The VN/1203 and 1918 viruses differentially regulate key cellular response pathways.
We next used Ingenuity Pathways Analysis to identify functional categories of differentially expressed genes. Our analyses showed that many of these genes were related to the inflammatory response (Fig. ) and included the upregulation of inflammasome genes in VN/1203-infected mice (but not in 1918-infected mice) at day 1 p.i. The upregulation of inflammasome genes in VN/1203-infected mice is also illustrated in the functional network shown in Fig. . In this network, genes depicted in blue were upregulated by VN/1203 but not by the 1918 virus, whereas genes depicted in orange were upregulated by VN/1203 virus but downregulated by the 1918 virus. Of particular note, the key inflammasome components CASP1 (caspase 1), interleukin 1β (IL-1β), and NLRP3 (nucleotide-binding domain and leucine-rich-repeat-containing protein 3) were upregulated in response to VN/1203 infection.
FIG. 3. VN/1203 virus differentially regulates the expression of inflammatory response genes. (A) Heat map illustrating ANOVA results for 142 inflammatory genes differentially transcribed within cutoff values of ≥2-fold change and ANOVA P ≤ 0.01 (more ...)
In addition, VN/1203 also upregulated the expression of tumor necrosis factor alpha (TNF-α), a potent inflammatory molecule; IFN-γ; eukaryotic initiation factor 2 AK2 (eIF2AK2); protein kinase RNA activated (PKR); and additional chemokines and inflammation-related genes. Quantitative RT-PCR analyses were performed to verify the expression of specific transcripts, including IL-1β, NLRP3, CASP1, and TNF-α. The results from this method correlated well with the microarray results (see Fig. S3 in the supplemental material). Later during infection (days 3 and 4 p.i.), the inflammatory responses induced by the two viruses were still different but did not involve the inflammasome, and the numbers of genes differentially regulated were fewer than observed on day 1. This striking difference in the quality of the early inflammatory responses to two highly pathogenic influenza viruses correlates with the severity of disease.
We also observed that at day 1 p.i., VN/1203 infection induced the differential expression of genes associated with viral sensing, neutrophil activation, NF-κB signaling, and chemokine signaling (Fig. ). Quantitative RT-PCR analyses were performed to verify the expression of specific transcripts, including IFN-β1 and IFN-γ. The results from this method correlated well with the microarray results (see Fig. S4A and B in the supplemental material). Because the products of many of these genes play important roles in regulating cellular gene expression, the activation of these genes may also be associated with the earlier and more robust host transcriptional and inflammatory response observed in VN/1203-infected animals.
FIG. 4. The VN/1203 and 1918 viruses differentially regulate key cellular signaling pathways. Shown is ANOVA of microarray data from lung samples using cutoff values of ≥2-fold change and ANOVA P ≤ 0.01 comparing the infections of wild-type animals (more ...) VN/1203 dissemination correlates with sustained activation of the inflammatory response and altered hematological function and lipoxin signaling.
To attempt to gain insight into the ability of VN/1203 to disseminate to the brain and spleen, we also devised an analysis strategy to identify gene expression changes that correlated with tissue dissemination. Briefly, gene expression data from all time points were stratified into four groups: wild-type mice infected by the 1918 virus, wild-type mice infected by VN/1203, IFNR1−/−
mice infected by the 1918 virus, and IFNR1−/−
mice infected by VN/1203. Among these groups, only wild-type mice infected by the 1918 virus showed no dissemination during infection. Differentially expressed genes in each group compared to mock infection were identified (≥2.0-fold change), and pathway analysis was carried out using Ingenuity Pathway Analysis and GeneGo MetaCore. Two functional categories, which included genes associated with hematological development and function and genes associated with the inhibitory effects of lipoxin on inflammation, leukocyte trafficking, and neutrophil-mediated tissue injury, showed statistically significant changes in the groups associated with virus dissemination but not the group not associated (wild-type mice infected by 1918). Many of the genes in the hematological development and function category are associated with proinflammatory responses and were strongly upregulated during VN/1203 infection (Fig. ). Lipoxins, in contrast, are eicosanoids with potent anti-inflammatory properties (37
). The gene responsible for lipoxin biogenesis, Alox5, was downregulated during VN/1203 infection (Fig. ), as was the gene encoding suppressor of cytokine signaling 2 (Socs2), the expression of which can be induced by lipoxins to control proinflammatory responses (22
). These finding suggest that not only does VN/1203 infection result in the induction of numerous proinflammatory genes, but lipoxin-mediated anti-inflammatory responses are also impaired. No change in the expression of Alox5, Socs2, or Fprl1 (the primary receptor for lipoxins) was observed in response to infection with the 1918 virus.
FIG. 5. Association of hematological function and lipoxin signaling with VN/1203 virus dissemination. (A) Differential regulation of hematological system development- and function-related genes. (B) Differential regulation of lipoxin signaling. Shown is ANOVA (more ...)
We also investigated the observed dissemination of the 1918 virus to brain and spleen during infection of IFNR1−/− mice. Our results indicated that the host responses elicited by 1918 virus infection were distinctly different in wild-type and immune-deficient animals. While the activation of important host functions, such as immune cell trafficking, inflammatory response, and cell death, were almost at similar levels in both mouse strains by day 1 p.i., these functions changed dramatically in wild-type mice, which responded to 1918 infection by an steady increase in the expression of genes associated with these functions by days 3 and 4 p.i. However, far fewer genes in these categories were transcriptionally activated in IFNR1−/− mice by days 3 and 4 p.i. (see Fig. S5 in the supplemental material).
VN/1203 and 1918 viruses induce the activation of type I IFN-regulated genes even in the absence of the IFN-α/β receptor.
Activation of IFN signaling is an important component of the innate antiviral response, and our previous studies of the 1918 virus in mice and VN/1203 in macaques suggested differences in IFN signaling in response to these viruses (6
). We therefore sought to directly compare the IFN response to these viruses in the mouse infection model. To identify IFN-regulated genes, we treated uninfected mice with IFN and identified the resulting differential expression of genes in the lungs. We also took advantage of mouse genetics and used IFNR1−/−
mice to determine the importance of an intact IFN response to the host response to these viruses.
To identify IFN-regulated genes, we treated wild-type or IFNR1−/−
mice with 10,000 U of recombinant human IFN-α A/D and harvested lung tissue at 8 and 24 h after treatment. These samples were then used to generate a set of 854 type I IFN-regulated genes, which was used as a common gene set for clustering and analysis of variance (ANOVA) of gene expression data from virus-infected animals. We found that activation of type I IFN-regulated genes was stronger in VN/1203-infected mice than in mice infected with the 1918 virus and that VN/1203 elicited steady and sustained activation of these genes even in animals lacking the IFN-α/β receptor (Fig. ). In animals infected with the 1918 virus, there was little activation of the IFN-regulated genes at day 1 p.i., but by days 3 and 4, IFN-regulated gene expression reached almost the same level as that induced by VN/1203. Since it was possible that IFNR1−/−
mice compensated for the absence of IFNR1 with increased expression of IFN-λ3 (also known as IL28B) (4
), we used quantitative RT-PCR to detect IL28B in lung samples at 1, 3, and 4 days p.i. We found that IL28B was not overexpressed in the IFNR1−/−
mice and that expression correlated with the temporal regulation of IFN-β1 and, to a lesser extent, IFN-γ (see Fig. S4C in the supplemental material).
FIG. 6. Expression of interferon-regulated genes in response to interferon treatment or VN/1203 or 1918 infection. (A) Expression of type I interferon response genes in wild-type or IFNR1−/− mice infected with 1918 and VN/1203 viruses. Expression (more ...)
IFNR1−/− mice infected with either virus died sooner than infected wild-type animals, and IFNR1−/− mice infected with VN/1203 died approximately 3 days sooner than those infected with the 1918 virus (see Fig. S6A in the supplemental material). The percentage of survival was determined by the weight loss parameter, according to the CDC's Institutional Animal Care and Use Committee (see Fig. S1B in the supplemental material). VN/1203 disseminated to brain, liver, and spleen, and in contrast to wild-type animals, the 1918 virus was also detected in the brains and spleens of IFNR1−/− mice at day 5 p.i. (see Fig. S6B in the supplemental material). As in the case of wild-type mice, viral dissemination in the IFNR1−/− mice did not correlate with the viral titer (see Fig. S6C in the supplemental material). Even though the 1918 virus was detected in the brains and spleens of IFNR1−/− mice at day 5 p.i., histopathology revealed no lesions in these organs (Fig. ). Lesions in spleens were observed in VN/1203-infected IFNR1−/− mice (Fig. ). Staining for influenza virus antigen in the bronchus was more uniformly distributed in VN/1203-infected than 1918-infected mice, and staining was more uniform in bronchiolar epithelium than in alveolar cells (Fig. ).
FIG. 7. Histopathology analysis of 1918 and VN/1203 infections in IFNR1−/− mice. (A) Normal spleen of an IFNR1−/− mouse infected by 1918 virus at day 1 p.i. (B) Normal liver of an IFNR1−/− mouse infected by 1918 (more ...)
To determine whether we could recapitulate our results using a simpler system, we performed in vitro 1918 and VN/1203 infection experiments using MEFs from wild-type and IFNR1−/− mice. Cells were harvested at 8 and 24 h p.i. for microarray analysis. Our analyses showed that wild-type and IFNR1−/− MEFs both exhibited a type I IFN response to VN/1203 infection, whereas the 1918 virus elicited weak activation of IFN-regulated genes. Interestingly, 1918 infection of MEFs recapitulated the delayed activation of the type I IFN response that was observed in mouse lungs (Fig. ). These results clearly indicate a significant difference in the host responses to the H1N1 1918 virus and the avian H5N1 VN/1203 virus and correlate well with our mouse microarray data. The combination of stronger inflammatory and type I IFN responses induced by VN/1203 virus likely contributes to the increased pathogenesis of this virus in the mouse model.