1 Department of Infectious Diseases (Division of Virology), St Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 381052794, USA
2 Department of Immunology, St Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 381052794, USA
3 Department of Pathology, St Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 381052794, USA
4 Department of Pathology, University of Tennessee, Memphis, TN 38163, USA
5 Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia
Correspondence
Robert G. Webster
robert.webster{at}stjude.org
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ABSTRACT |
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Present address: Department of Pediatrics, Steele Memorial Children's Research Center, University of Arizona, Tucson, AZ, USA.
Present address: Department of Veterinary Medicine, University of Maryland, 8075 Greenmead Drive, College Park, MD 20742-3711, USA.
Present address: Department of Microbiology, The University of Tennessee, Walters Life Sciences F419, 1414 W. Cumberland Avenue, Knoxville, TN 37996, USA.
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INTRODUCTION |
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In the lungs and spleens of infected mice, all of the H5N1/97 viruses isolated from humans induce a high concentration of cytokines, such as interleukin (IL)-1 and interferon (IFN)-
, and of the chemokine macrophage inflammatory protein (MIP)-1
, (Tumpey et al., 2000
). In human primary monocyte-derived macrophages, the H5N1/97 viruses induced much higher transcription of pro-inflammatory cytokines, particularly tumour necrosis factor (TNF)-
and IFN-
, than did H3N2 or H1N1 viruses (Cheung et al., 2002
). The non-structural (NS) gene segment of the H5N1/97 viruses played a role in the increased TNF-
transcription (Cheung et al., 2002
). In pig lung epithelial cells (Seo et al., 2001
), the NS gene of H5N1/97 viruses was shown to confer resistance to the antiviral effects of IFNs and TNF-
(Seo et al., 2002
), and TNF-
was shown to exert powerful anti-influenza virus effects (Seo & Webster, 2002
). Further, when this gene was inserted by reverse genetics into the non-pathogenic A/Puerto Rico/8/34 (H1N1) laboratory strain, it conferred increased pathogenicity in pigs (Seo et al., 2002
). These effects required the presence of Glu at position 92 in the non-structural protein 1 (NS1) (Seo et al., 2002
), a residue unique to H5N1/97 viruses. These characteristics were proposed to contribute to the unusual severity of human H5N1 disease (Cheung et al., 2002
; Seo et al., 2002
). However, the influence of the NS gene of H5N1/97 viruses on molecular pathogenesis and host immune response remains unclear.
Here, we report the influence of the Hong Kong H5N1 influenza viruses' NS genes on viral pathogenicity in a mouse model. We used reverse genetics to create H1N1 A/Puerto Rico/8/34 reassortants containing the NS gene of a human 1997 H5N1 isolate, the NS gene of an avian 2001 H5N1 virus isolate, and an altered form of the NS gene of the human 1997 H5N1 isolate encoding a Glu92Asp substitution in NS1. The avian virus was one of the genotypes isolated during a 2001 outbreak in the Hong Kong poultry markets (Guan et al., 2002
), whose NS genes encode an NS1 protein with a unique deletion of 5 aa. A similar NS gene was identified in H5N1 virus isolated from two human cases, one of which was fatal in Hong Kong in February 2003 (Guan et al., 2004
). We determined the pathogenicity, organ tropism and pulmonary replication kinetics of the NS gene-reassortant viruses in mice and characterized the influence of the NS gene on inflammatory response (induction of cytokines in the lungs) and specific B- and T-cell responses. We observed an imbalance in the level of pro-inflammatory and anti-inflammatory cytokines induced by virus carrying the NS gene of H5N1/97 influenza viruses; this imbalance may contribute to the unusual severity of disease caused by the H5N1/97 influenza viruses.
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METHODS |
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Madin-Darby canine kidney (MDCK) cells and 293T human embryonic kidney cells were used for rescue of reassortant viruses from plasmids. MDCK cells were cultured in minimal essential medium (MEM) supplemented with 10 % fetal bovine serum (FBS). 293T cells were cultured in Opti-MEM I (Life Technologies) containing 5 % FBS.
Isolation of viral RNA, reverse transcription, PCR amplification and sequencing.
Extraction of viral RNA, synthesis of cDNA and PCR were performed as described previously (Hoffmann et al., 2001), with minor modifications. Sequencing was performed by the Hartwell Center for Biotechnology at St. Jude Children's Research Hospital with the rhodamine dye-terminator cycle sequencing Ready Reaction kit with AmpliTaq DNA polymerase FS (Perkin-Elmer Applied Biosystems).
Cloning and generation of viruses by reverse genetics.
The eight genes of PR/8 and the NS genes of HK/156/97 and Ck/HK/YU562/01 were cloned as previously described (Hoffmann et al., 2000; Hoffmann et al., 2001
; Hoffmann et al., 2002
). Plasmids were sequenced as described above, and the sequences were compared with those generated from the wild-type virus. Only clones that exactly matched the parental virus sequence were used for virus rescue by reverse genetics. Viruses were rescued by using the eight-plasmid system (Hoffmann et al., 2000
), with minor modifications. Briefly, eight plasmids (1 µg of each) were incubated for 45 min with Trans-LTI (Panvera) in Opti-MEM I and were used to transfect a 1 : 1 mixture of 293T and MDCK cells as described previously (Hoffmann et al., 2000
). Supernatant collected from transfected cells after 72 h was used to inoculate 10-day-old embryonated chicken eggs. PR/8 virus and the following NS-gene reassortant viruses were rescued from the plasmids: PR/8xNS HK/156/97 (PR/8xNS H5N1/97), PR/8xNS Ck/HK/YU562/01 (PR/8xNS H5N1/01) and PR/8xNS1E92
D HK/156/97 (PR/8xNS mut. H5N1/97). Allantoic fluid containing virus was harvested, and its infectivity was titrated in eggs; virus titres were expressed as log10 of the 50 % egg infective dose per 0·1 ml of fluid (log10 EID50 per 0·1 ml), according to the method of Reed and Muench (1938)
. Virus stocks were divided into aliquots and stored at 80 °C.
Site-directed mutagenesis.
We used PCR with overlapping internal primers (Seo et al., 2002) to substitute Asp for Glu at position 92 of the HK/156/97 virus NS1 protein. The reassortant virus that incorporated this gene in the background of the PR/8 virus was rescued as described above.
Mice and virus inoculation.
Female, 6-week-old BALB/c mice (The Jackson Laboratory) were used for studies of viral pathogenicity and determination of the mouse 50 % lethal dose (MLD50). Female, 10-week-old C57BL/6J mice (The Jackson Laboratory) were used for immunological experiments. To determine the MLD50, we anaesthetized mice in groups of four by isoflurane inhalation and infected them intranasally with 50 µl 10-fold serial dilutions of allantoic fluid in PBS. Animals were observed daily for 15 days for mortality. MLD50 was calculated by the method of Reed and Muench (1938) and was defined as the number of EID50 resulting in 50 % mortality. Viral pathogenicity (pulmonary viral titres, cytokine assays and histopathology) was studied in groups of 26 mice inoculated as described above with 50 µl PBS-diluted allantoic fluid containing 100 EID50 of virus per mouse. Three to six mice in each group were killed on days 3, 6, 7, 8, 9, 10 and 11 after inoculation, and lungs, brain and spleen were removed; an approximately 10 % homogenate of each tissue was prepared as described previously (Lipatov et al., 2003
) and stored at 80 °C. Virus was titrated in lungs, brain and spleen, and cytokine concentrations were determined in lungs. Virus in tissue homogenates was titrated and the EID50 was determined in 10-day-old embryonated chicken eggs. The lower limit of virus detection was 0·1 log10 EID50 per 0·1 ml of tissue homogenate.
For the immunological studies, groups of 10 C57BL/6J mice were inoculated intranasally with 100 EID50 of virus as previously described (Sarawar & Doherty, 1994). Animals were killed on days 7 and 10 after virus inoculation; the cervical lymph nodes (CLN), mediastinal lymph nodes (MLN) and spleen were removed, and blood and broncho-alveolar lavage (BAL) specimens were obtained.
Assay of cytokines and chemokine CXCL1 (KC).
Cytokine and chemokine proteins were measured in mouse lung homogenates collected on days 3, 6 and 9 after virus inoculation by using the Bio-Plex Mouse Cytokine 17-Plex panel (Bio-Rad Laboratories). Lung homogenate was clarified by centrifugation at 2000 g for 10 min. The assay was performed in 50 µl homogenate as specified by the manufacturer and was read on the Bio-Plex Protein Array system (Bio-Rad Laboratories).
Assay of virus-specific antibody-forming cells and antibodies.
Influenza-specific antibody-forming cells (AFC) were enumerated by ELISpot assay. Multiscreen HA 96-well filtration plates (Millipore) were coated with purified PR/8 influenza virus, and single-cell suspensions were plated and incubated as described previously (Sangster et al., 2000). Alkaline phosphatase-conjugated goat anti-mouse antibodies (Abs) specific for IgM, IgG1, IgG2b, IgG2c, IgG3 or IgA (Southern Biotechnology Associates) were diluted 1 : 500 in PBS plus 5 % BSA and added to the plates. After overnight incubation at 4 °C followed by extensive washing, the plates were developed at room temperature with 1 mg 5-bromo-4-chloro-3-indolyl phosphate (Sigma) ml1 in diethanolamine buffer and washed and dried. Spots representing individual AFC were counted on an Olympus SZH Stereozoom microscope. Negative control plates were coated with purified Sendai virus. No spots were seen when lymph node cell suspensions from naive mice were tested on plates coated with either influenza virus or Sendai virus.
Influenza-specific serum Abs were measured by ELISA (Sangster et al., 2000), using plates coated with 0·5 µg per well of purified, detergent-disrupted PR/8 influenza virus. Briefly, threefold serial dilutions of sample were incubated in the plates. Bound Ab was detected with alkaline phosphatase-conjugated goat anti-mouse Abs specific for IgM, IgG1, IgG2b, IgG2c, IgG3 or IgA (Southern Biotechnology Associates), and colour was developed with p-nitrophenylphosphate substrate (Sigma). Absorbance was read at 405 nm on a SpectraMax 340 microplate reader with SoftMax Pro software (Molecular Devices). The virus-specific Ab titre was defined as the reciprocal of the highest serum dilution giving an absorbance value greater than twice that of the samples from naive mice that were titrated in parallel.
Flow-cytometric analysis.
The kinetics and magnitude of the virus-specific CD8+ T-cell responses were analysed by flow cytometry. CD8+ T cells in the disrupted splenic tissue were enriched (Hou et al., 1994) by incubation with mAbs to CD4 (GK1.5) and MHC class II glycoprotein (M5/114.15.2) and then with magnetic beads coated with anti-rat and anti-mouse IgG (Dynal). Lymphocytes were isolated from the mouse lungs by BAL, and macrophages were removed by incubation on plastic for 1 h at 37 °C. The DbNP366 and DbPA224 tetramers (Altman et al., 1996
) were made by formation of a complex comprising H2Db plus the immunogenic NP366 (ASNENMETM) (Townsend et al., 1986
) or PA224 (SSLENFRAYV) (Belz et al., 2000
) peptides or H2Kb plus the polymerase 1 (PB1703, SSYRRVPGI) peptide (Belz et al., 2001
). The lymphocytes were incubated for 60 min at room temperature with the PE-conjugated tetramers in PBS/BSA/azide (Flynn et al., 1998
) and then stained with anti-CD8
-PerCPCy5.5 (PharMingen). Cells were sorted on a Becton Dickinson FACScan instrument, and data were analysed by using CELLQUEST software (Becton Dickinson Immunocytometry Systems).
Histopathologic analysis.
The lungs of mice infected with 100 EID50 of virus were harvested on day 6 after inoculation, washed in PBS, fixed in 10 % neutral buffered formalin and embedded in paraffin wax. Sections (5 µm) were stained with haematoxylin and eosin and microscopically reviewed.
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RESULTS |
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To determine whether the NS genes of H5N1 viruses influence the organ tropism of reassortant viruses, we identified and titrated virus in the lungs, brains and spleens of infected mice. No differences were observed in organ tropism between the PR/8 and NS reassortant viruses: all studied viruses only replicated efficiently in the lungs. No viruses were detected in the brains or spleens of mice killed on days 211 after inoculation or in those of mice that died during MLD50 titration. Fig. 1 shows the kinetics of virus replication in the lungs of mice inoculated with 100 EID50 of PR/8 virus or NS reassortants. PR/8 virus replicated to high titres in mouse lungs on days 37 after inoculation and was completely cleared from the lungs on day 9. Reassortant PR/8xNS H5N1/97 had titres only slightly higher than PR/8, but a significant quantity of infectious PR/8xNS H5N1/97 virus remained on day 9 after inoculation, and virus was not cleared from the lungs until day 11. Titres of PR/8xNS mut. H5N1/97 virus approximated those of PR/8 only at day 3. On days 68 after inoculation, the kinetics of replication resembled that of PR/8xNS H5N1/01 reassortant: both viruses were cleared from the lungs on day 9, and their titres were 1·02·5 log10 lower than those of PR/8 and PR/8xNS H5N1/97 (Fig. 1
).
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Cytokine and chemokine assays in the lungs of infected mice
We measured the concentration of cytokines and chemokine CXCL1 (KC) in the lungs of infected mice killed on days 3, 6 and 9 after inoculation. Mice inoculated with 50 µl sterile PBS served as negative controls. Eight cytokines and chemokines showed measurable concentrations greater than those of the controls. Fig. 2 shows the most significant differences among the viruses studied. Viruses that were highly pathogenic to mice, i.e. the original PR/8 and reassortant PR/8xNS H5N1/97, induced much higher levels of inflammatory cytokines and chemokine CXCL1 in infected lungs than did the reassortant viruses with low mouse pathogenicity, PR/8xNS mut. H5N1/97 and PR/8xNS H5N1/01. Further, pulmonary concentrations of IL1
, IL1
, IL6, granulocyte macrophage colony stimulating factor (GM-CSF), and IL8-like chemokine CXCL1 (keratinocyte-derived chemokine, KC) were clearly increased in mice infected with the reassortant PR/8xNS H5N1/97 as compared with mice infected with PR/8 (Fig. 2a, b, c, g, h
). The reassortant PR/8xNS mut. H5N1/97 induced lower levels of these cytokines and chemokine KC than did PR/8, and concentrations of IL1
, IL1
, IL6, GM-CSF and KC were close to negligible in the lungs of mice infected with PR/8xNS H5N1/01 viruses (Fig. 2a, b, c, g, h
). Levels of IL12 and IFN-
were similar in the lungs of mice infected with the pathogenic viruses PR/8 and PR/8xNS H5N1/97, were lower in the lungs of mice infected with PR/8xNS mut. H5N1/97, and were very low in mice infected with PR/8xNS H5N1/01 (Fig. 2e, f
). The NS-reassortant viruses demonstrated interesting differences in their induction of the anti-inflammatory cytokine IL10: in the lungs of mice infected with PR/8xNS H5N1/97, the concentration of this cytokine was only half that induced by PR/8, whereas the reassortant that contained the mutated NS gene, PR/8xNS mut. H5N1/97, increased the concentration of IL10 to a level 2·5 times that induced by PR/8xNS H5N1/97 (Fig. 2d
). A trace amount of IL10 was detected in the lungs of mice infected with PR/8xNS H5N1/01 virus (Fig. 2d
).
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Specific B- and T-cell responses
The enhanced virulence and delayed clearance of PR/8xNS H5N1/97 virus that we observed can be consistent with diminished humoral and T-cell responses. We investigated whether adaptive immune responses were modified by the NS genes of H5N1 viruses. The mean frequency of influenza-specific AFC in the MLN on day 10 demonstrated that PR/8 and PR/8xNS H5N1/97 elicited strong responses of similar magnitude (Fig. 3). Therefore, the delayed pulmonary clearance of PR/8xNS H5N1/97 was not caused by a deficient humoral response. The AFC response in the MLN was negligible on day 7 after inoculation with PR/8xNS H5N1/01 (data not shown) but had emerged fully on day 10 (Fig. 3
), perhaps reflecting low early virus titres in the lungs. Interestingly, the response to PR/8xNS mut. H5N1/97 on day 10 lacked the usually prominent IgA component; this finding may also be related to diminished replicative capability. The serum antibody concentrations determined by ELISA on days 7 and 10 after inoculation corresponded completely with the results of ELISpot assays (data not shown).
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DISCUSSION |
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Seo and co-authors (Seo et al., 2002) first showed that the NS gene of a human isolate of Hong Kong H5N1/97 viruses dramatically increased the pathogenicity of PR/8 virus in pigs, and that this effect required the presence of Glu at residue 92 of the NS1 protein. The observed effect was attributed to resistance to the antiviral effects of IFNs and TNF-
conferred by the NS gene of these viruses (Seo & Webster, 2002
; Seo et al., 2002
). In contrast, recent studies using primary human monocyte-derived macrophages as an in vitro model found that the NS genes of H5N1/97 viruses induce the transcription of TNF-
and IFN-
, a process that can contribute to pathogenesis (Cheung et al., 2002
).
The study presented here describes in detail the pathogenesis of infection caused by reassortant viruses containing the NS gene of H5N1/97 and H5N1/01 viruses in a mouse model, including the production of cytokines and chemokine CXCL1 in mouse lung and the characterization of adaptive immune responses. The NS1 protein encoded by the NS gene of the H5N1/01 avian viruses has a deletion of 5 aa (residues 7882) and confers resistance to the antiviral effects of TNF-, IFN-
and IFN-
in cell culture (Seo et al., 2002
), but the pathogenicity of reassortant virus carrying this gene had not been studied in vivo. Because the H5N1 viruses isolated from two human cases in Hong Kong in 2003 have a similar NS gene (Guan et al., 2004
), we included the gene in this study.
Our results demonstrated that the NS gene of H5N1/97 viruses confers high pathogenicity in mice when inserted into the PR/8 background. PR/8xNS H5N1/97 replicated efficiently in mouse lung, and clearance of this reassortant required 2 days longer than clearance of PR/8. In mice, as previously observed in swine, the pathogenic characteristics conferred by the NS gene originating from the H5N1/97 viruses required the presence of Glu at residue 92 of the NS1 protein. Reassortant virus with a Glu92Asp mutation in NS1 caused a lower rate of mortality and lower mean pulmonary virus titres. These results demonstrate that the mouse model is suitable for studies of the pathologic effects caused by the NS gene of H5N1/97.
Surprisingly, the NS gene of H5N1/01 origin completely attenuated the pathogenicity of PR/8 virus in mice. Because this reassortant was resistant to the antiviral effects of IFNs and TNF- in pig lung epithelial cells (Seo et al., 2002
), we examined its pathogenicity in Yucatan miniature pigs, using PR/8xNS H5N1/97 reassortant as a positive control virus. In this preliminary experiment, PR/8xNS H5N1/01 virus replicated in the upper respiratory tract to lower titres than PR/8xNS H5N1/97 and caused no signs of disease, whereas infection with PR/8xNS H5N1/97 virus resulted in serious illness (data not shown). Therefore, despite the similar properties conferred upon PR/8 by the NS genes of H5N1/97 and H5N1/01 viruses in cell culture experiments, PR/8 reassortants with these genes exert opposite pathological effects in vivo, in both mouse and pig models. This finding suggests that the resistance to the antiviral effects of IFNs and TNF-
conferred by the NS genes of H5N1/97 and H5N1/01 viruses is not the main mechanism of the high virulence associated with the NS gene of H5N1/97 origin. The attenuated pathogenicity of PR/8xNS H5N1/01 reassortant virus also raises questions as to whether the NS gene, which encodes the NS1 protein with a 5 aa deletion, similar to that observed in human H5N1/03 lethal virus isolates, can contribute to high virulence in humans? Mouse and pig models do not reflect exactly the pathogenicity of H5N1 viruses in humans. However, it is possible to propose that in the case of H5N1/03 human isolates high pathogenicity can be determined by other viral genes or gene constellations. To find an answer, additional studies of the influence of H5N1/01 and H5N1/03 NS genes in virulence are required including studies on human primary cells and in primate models.
To explore other possible explanations for the pathogenicity of the PR/8xNS H5N1/97 reassortant virus, we assayed cytokines and IL8-like chemokine CXCL1 (KC) in the lungs of infected mice. Primary influenza A virus pneumonia in mice is known to induce the production of cytokines and chemokines in lung tissues (Hennet et al., 1992; Monteiro et al., 1998
; Sarawar & Doherty, 1994
). The cytokines have been proposed to contribute to the recruitment and activation of virus-specific T cells and the induction and development of immune response (Doherty et al., 1992
; Hennet et al., 1992
; Sarawar & Doherty, 1994
). On the other hand, elevated levels of inflammatory cytokines are also associated with acute respiratory distress syndrome, multiple organ dysfunction (Headley et al., 1997
) and haemophagocytic syndrome (Fisman, 2000
) in humans. Increased levels of inflammatory cytokines, especially IL6, were associated with pathological signs in human volunteers experimentally infected with influenza viruses (Hayden et al., 1998
; Kaiser et al., 2001
; Skoner et al., 1999
). Moreover, the full post-mortem reports available for two cases of H5N1/97 virus-induced pneumonia describe reactive haemophagocytic syndrome with increased concentrations of IL6, TNF-
and IFN-
(To et al., 2001
).
Our assays of a panel of cytokines in the lungs of infected mice showed that infection with reassortant virus bearing the NS gene of H5N1/97 induces high concentrations of the inflammatory cytokines IL1, IL1
, IL6 and IL8-like chemokine (KC); concentrations were higher even than those induced by parental, pathogenic PR/8 virus. This characteristic was related to the presence of Glu at residue 92 of the encoded NS1 protein. Reassortant virus with a mutated NS1 gene encoding Asp at position 92 induced significantly lower levels of these inflammatory cytokines and chemokines. On the other hand, PR/8xNS mut. H5N1/97 virus induced a higher level of the anti-inflammatory cytokine IL10 in infected mouse lungs than did the other tested viruses. This cytokine was also elevated in the lungs of mice infected with PR/8 virus, whereas it was decreased in the lungs of animals infected with PR/8xNS H5N1/97 virus. IL10 is known to suppress the transcription of lipopolysaccharide-induced inflammatory cytokines (Hamilton et al., 2002
; Moore et al., 2001
). Thus, the lungs of mice infected with PR/8xNS H5N1/97 demonstrated elevated levels of pro-inflammatory cytokines and chemokine, particularly IL6 and KC, and a low level of the anti-inflammatory cytokine IL10. The high level of inflammatory cytokines is also consistent with the histological findings: the pulmonary lesions in mice infected with PR/8 and PR/8xNS H5N1/97 were similar in character, but the inflammation and cellular infiltration were more intense in the latter mice.
Primary infection of mice with the reassortant viruses, as well as with PR/8 virus, induced normal, non-deficient humoral and virus-specific T-cell responses. The enhanced virulence and delayed clearance of PR/8xNS H5N1/97 virus is not consistent with diminished humoral and T-cell responses. In fact, a greater magnitude of CD8+ T-cell response was induced by the more pathogenic strains. Some observations, particularly the very low number of CD8+ cells in the spleen and BAL specimens of mice infected with PR/8xNS mut. H5N1/97, could be both a cause and the effect of the attenuated pathogenicity and replication of this virus. Infection with PR/8xNS H5N1/97 induced a large CD8+ T-cell response, especially in infected lungs. It is possible that this characteristic of the H5N1/97 NS gene requires the presence of Glu at position 92 of the encoded NS1 protein and is lost in virus with a mutated NS gene.
Our findings support two main conclusions. First, the NS gene of Hong Kong H5N1/97 viruses can generate and support high pathogenicity when inserted into a background virus that is pathogenic (like PR/8 in mice) or non-pathogenic (like PR/8 in pigs). The pathogenicity conferred by the H5N1/97 NS gene may be caused by a cytokine imbalance; that is, by elevated production of inflammatory cytokines and chemokine CXCL1 and the simultaneous decreased production of anti-inflammatory cytokine IL10. These functional properties of the H5N1/97 NS gene require the presence of Glu at residue 92 of the encoded NS1 protein. This residue has been identified only in the NS1 protein of Hong Kong H5N1/97 human isolates and phylogenetically closely related avian viruses. The cytokine imbalance caused by the virus bearing the H5N1/97 NS gene is consistent with the detailed post-mortem report on two humans who died in Hong Kong in 1997 of H5N1 pneumonia and explains, at least partially, the unusual severity of H5N1/97 influenza virus infection.
Second, the detailed pathological and immunological studies reported here show that the mouse model is useful for further dissection of the molecular mechanisms and pathways underlying the high pathogenicity conferred by the unique NS gene of H5N1/97 influenza virus origin.
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ACKNOWLEDGEMENTS |
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Received 5 October 2004;
accepted 10 December 2004.