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Short Communication |
1 Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan
2 Department of Pediatrics, Nagoya Ekisaikai Hospital, Nagoya, Japan
3 Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Japan
4 Development Center for Targeted and Minimally Invasive Diagnosis and Treatment, Fujita Health University, Toyoake, Japan
5 Department of Pediatrics, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
Correspondence
Hiroshi Kimura
hkimura{at}med.nagoya-u.ac.jp
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ABSTRACT |
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Details of genes up- and downregulated during the acute phase of infection are available as supplementary material in JGV Online.
Present address: Department of Virology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. 
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; Wright & Webster, 2001). The host response to influenza virus has not been fully elucidated in humans. Influenza virus replicates in the epithelial cells of the upper respiratory tract, which has several mechanisms to protect against influenza infection. Patients with influenza have respiratory symptoms, such as cough and rhinorrhoea, owing to mucosal inflammation and an influx of polymorphonuclear cells (Wright & Webster, 2001). In addition to these symptoms, most patients with influenza suffer from systemic symptoms, including high fever, myalgias and malaise. To date, the initial responses to influenza virus infection have been investigated mainly in the respiratory tract (Hayden et al., 1998; Kaiser et al., 2001; Skoner et al., 1999); however, the global host response to the virus has not been investigated in humans.
Recently, there has been an increase in the number of reports of influenza-associated encephalopathy in Japan and other countries (Kasai et al., 2000; Morishima et al., 2002; Newland et al., 2003). Patients with influenza-associated encephalopathy often develop multi-organ failure and have high rates of morbidity and mortality. In addition to encephalopathy, influenza virus infection is associated with a higher incidence of febrile convulsions compared with other viral infections (Chiu et al., 2001; Kawada et al., 2004). The pathogenesis of these complications has not been fully understood, although it may be that encephalopathy and febrile convulsion are provoked by the same mechanism (Kawada et al., 2003b).
The aim of the present study was to analyse gene-expression profiles in the peripheral blood of influenza virus-infected patients in order to reveal the mechanisms of the host response to influenza virus. An oligonucleotide microarray was used to reveal global host responses to influenza virus infection in children. This technique allows the expression patterns of thousands of genes to be studied in parallel. In addition, the gene-expression profiles of influenza patients with or without febrile convulsion were compared.
Nine patients with influenza who presented during one of two influenza seasons (2002–2004 seasons) were enrolled in this study. Their ages ranged from 3 months to 4 years (median 2 years) and all had typical clinical signs and symptoms of influenza, such as acute onset of fever, cough, chills and nasal congestion. Five patients with febrile convulsion were included. They had convulsions of short duration (1–5 min) provoked by fever but recovered without any sequelae. Four patients without febrile convulsion from the same district as those with febrile convulsions were selected as age-matched controls. None of the patients had underlying disease. Cultures of blood, cerebrospinal fluid and throat swabs showed no indication of central nervous system infection by other viruses or bacteria. They also did not receive aspirin or folk remedies during the influenza episodes. Influenza infection was confirmed by a fourfold or greater increase in haemagglutinin inhibition test titres and/or virus antigen detection in the throat using the latex agglutination test. All viruses were identified as type A influenza (H3N2). At the time of enrolment, antibody titres against H3N2 were negative (<10) in six of seven tested patients, suggesting that most patients were naive to influenza virus. Only one patient had received influenza vaccination before infection. As controls, four healthy children were also enrolled in this study. All samples were obtained from Aichi prefecture, which is located in the centre of Japan. Informed consent was obtained from the parents of all children participating in this study. This study was approved by the ethical committee of Nagoya University Graduate School of Medicine.
Peripheral blood was collected from patients both in the acute phase of the disease [0–2 days after the patients had high-grade fever (>38.0 °C)] and in the early convalescent phase (1–5 days after body temperature returned to normal). Whole blood (2.5 ml) was drawn directly into a PAX gene blood RNA tube (Qiagen), and total RNA was purified with PAX gene blood RNA kits (Qiagen) according to the manufacturer's protocols. White blood cells in the acute phase of influenza contained a mean of 61 % granulocytes, 31 % lymphocytes and 5 % monocytes. Total RNA (400 ng) was amplified and labelled using the Agilent Low RNA Input fluorescent linear amplification kit (Agilent Technologies). For each patient, acute-phase and early convalescent-phase samples were labelled with Cy3 and Cy5, respectively. Hybridizations on the microarray slide of Human 1A (Agilent Technologies), which contains 17 086 sequenced human genes, were carried out for 17 h in a rotating hybridization oven using an in situ hybridization kit (Agilent Technologies). The slides were washed as indicated in the protocol and were then scanned with an Agilent Scanner (G2565).
Microarray expression data were obtained using the Feature Extraction software (Agilent Technologies). The raw data on the intensity of gene expression in the acute phase were normalized by Z transformation to obtain Z scores (Cheadle et al., 2003). The latter were calculated by subtracting the mean intensity of gene expression in the convalescent phase from the mean intensity of gene expression during the acute phase for each gene and then dividing that result by the standard deviation of gene expression intensity during the convalescent phase. Using this approach, it was possible to compare the results from different experiments. A gene was defined as being upregulated in the acute phase if the Z score was greater than 1.96 (P <0.05) and downregulated if the Z score was less than –1.96. In addition, levels of gene expression were compared among clinical categories and Z scores were calculated for each clinical category [no febrile convulsion, Z (N); febrile convulsion, Z (FC)]. Differences in gene expression were evaluated by comparing these scores. The transcription of a gene was considered to be upregulated more in patients with febrile convulsion than in patients with no febrile convulsion if Z (FC)–Z (N) was greater than 3.92 (P <0.05). The applicability of these methods to microarray data was examined and verified in previous studies (Hong et al., 2004; Kim et al., 2003).
The molecular changes associated with influenza virus infection in vivo were investigated by microarray analysis of RNA from peripheral whole-blood samples using a 17 086-element oligonucleotide microarray. Gene-expression intensity data collected from all nine patients with influenza infection were used. Overall, 200 genes were found to be upregulated (Z >1.96) and 20 genes were found to be downregulated (Z < –1.96) in acute-phase influenza. Fig. 1 shows the Z scores of 15 genes that were upregulated and 15 genes that were downregulated. Interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) and vipirin (cig5), which is also induced by interferon, were respectively the most and second most upregulated genes in acute-phase infection. Protein tyrosine phosphatase receptor type C (PTPRC), which was the third most upregulated gene, is an essential regulator of T- and B-cell antigen receptor signalling. Prolactin (PRL), which is a growth hormone that stimulates lactation, was the most downregulated gene in acute-phase influenza. Summaries of the genes that were up- or downregulated in the acute phase are shown in Supplementary Table S1 available in JGV Online. RNA from healthy children was also analysed using the same assay system. Of 200 genes that were upregulated in the acute phase of influenza, only three were upregulated (Z >1.96) in the control children (Supplementary Table S1). Similarly, of 20 genes that were downregulated in acute influenza, only two genes were downregulated (Supplementary Table S1). Thus, in the group of healthy children, only a few genes were up- or downregulated, indicating that the up- or downregulation seen in influenza patients is specific to influenza infection.
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). Each gene was classified according to its ontology, by which genes are organized into hierarchical categories based on biological process, molecular function and cellular component (Ashburner et al., 2000; Boyle et al., 2004). Based on an analysis using EASE software, several gene ontology categories were identified as being overrepresented (EASE score <0.05). Genes assigned to the category of immune response genes were the most overrepresented among the upregulated genes (EASE score 0.00000001). Genes in ontology categories associated with the immune response, such as defence response and response to biotic stimulus, were also overrepresented (EASE scores of 0.00004 and 0.00007, respectively). Representative gene ontology categories considered to be overrepresented are shown in Fig. 2.
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. By contrast, five genes were found to be downregulated in patients with febrile convulsion [Z (FC) – Z (N) < –3.92]; these are also listed in Table 1. Gene ontology analysis was also carried out for upregulated or downregulated genes in patients with febrile convulsion, but no gene ontology categories were found to be significantly overrepresented (EASE score >0.05).
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; Tong et al., 2004). Furthermore, the host immune response to influenza A virus was investigated using the lungs of influenza-infected mice and pigtailed macaques (Baskin et al., 2004; Kash et al., 2004). These in vitro and animal studies showed that influenza virus infection results in a significant induction of genes involved in the interferon pathway. To investigate the global host response to influenza, gene-expression profiling of peripheral blood from patients with influenza was carried out using a microarray. We showed that interferon-regulated genes, such as IFIT1, IFIT2 and vipirin, were strongly upregulated in the acute phase of influenza. Furthermore, many other genes associated with the immune response were upregulated. These results are consistent with the gene-expression profiles obtained by microarray analysis of influenza virus-infected lung cells (Geiss et al., 2002; Tong et al., 2004). It has been shown that interferon-regulated genes are activated in vitro in human peripheral blood infected with influenza virus (Ronni et al., 1995). Our results are compatible with these gene-expression profiles, indicating that our method is a reliable approach to investigate the pathogenesis of influenza. The global host response to influenza virus could be established in clinical samples.
The clinical manifestations of influenza in children are similar to those seen in adults, but there are also some distinct differences between these two populations. In children, influenza is accompanied occasionally by febrile convulsion and rarely by encephalopathy (Chiu et al., 2001; Morishima et al., 2002). Influenza-associated encephalopathy is a severe disease with high mortality. Febrile convulsion, which results in impaired consciousness lasting <24 h, may be difficult to differentiate from encephalopathy, and it may be that encephalopathy and febrile convulsion are provoked by the same mechanisms (Kawada et al., 2003b). In this study, 22 genes were upregulated and five genes were downregulated in patients with febrile convulsion compared with their expression in patients without febrile convulsion. Although gene ontology analysis did not identify significantly overrepresented categories for these genes, we found that some interesting genes were included. Apolipoprotein L (APOL1) and ATP-binding cassette, subfamily D, member 2 (ABCD2) are associated with fatty acid metabolism (Duchateau et al., 1997; Holzinger et al., 1999). This finding is of interest because Reye's syndrome, which involves acute encephalopathy, is associated with a disturbance in fatty acid metabolism (Ogburn et al., 1982) and often follows influenza virus infection and salicylate therapy in children (Belay et al., 1999; Starko et al., 1980). None of the patients enrolled in this study received salicylate, but it may be that patients with genetic abnormalities in fatty acid metabolism are more likely to experience neurological complications. ABCD2 is also an adrenoleukodystrophy-related protein. Adrenoleukodystrophy is characterized by demyelination in the central nervous system (Moser, 2000). Furthermore, neureglin 1 (NRG1), which induces the growth and differentiation of neuronal and glial cells (Law et al., 2004), was also upregulated.
The pathogenesis of influenza-associated encephalopathy remains unclear. Viral RNA is rarely detected in the cerebrospinal fluid, and viral antigen is not present in the brain (Ito et al., 1999; Kawada et al., 2003a). Pathological findings, including the lack of detectable viral antigen and inflammatory cells in brain tissues, suggest that direct viral invasion and inflammation are therefore unlikely to be the cause of this disease. Instead, several studies have revealed that serum concentrations of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and IL-1
, are elevated and are related to the clinical severity of the encephalopathy (Aiba et al., 2001; Ito et al., 1999). In a previous study, we showed that transcription of IL-6 and IL-10 genes in peripheral leukocytes is upregulated in patients with influenza-associated encephalopathy (Kawada et al., 2003b). Furthermore, several studies have suggested an association between febrile convulsion and pro-inflammatory cytokines (Straussberg et al., 2001; Virta et al., 2002). In this study, the transcription levels of pro-inflammatory cytokine genes in patients with febrile convulsion were not significantly different from those in patients without febrile convulsion. Several reasons may account for this discrepancy. Firstly, unlike in patients with influenza-associated encephalopathy, the transcription levels of pro-inflammatory cytokines may not be upregulated in patients with febrile convulsion. Febrile convulsions might not be an appropriate marker of neurological complications and our results may not be generalized to other forms of neurological complications, such as Reye's syndrome, acute necrotizing encephalopathy and myelitis. Secondly, it may be that the differences in the transcription levels were too small to be detected by microarray techniques.
To our knowledge, this is the first study to show the global host responses in peripheral blood of patients with influenza. The data suggest that microarrays can be applied to the investigation of clinical samples from patients with acute viral infection. Our results should help to clarify the pathogenesis of influenza and its neurological complications, including encephalopathy.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Ashburner, M., Ball, C. A., Blake, J. A. & 17 other authors (2000). Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25, 25–29.[CrossRef][Medline]
Baskin, C. R., Garcia-Sastre, A., Tumpey, T. M., Bielefeldt-Ohmann, H., Carter, V. S., Nistal-Villan, E. & Katze, M. G. (2004). Integration of clinical data, pathology, and cDNA microarrays in influenza virus-infected pigtailed macaques (Macaca nemestrina). J Virol 78, 10420–10432.
Belay, E. D., Bresee, J. S., Holman, R. C., Khan, A. S., Shahriari, A. & Schonberger, L. B. (1999). Reye's syndrome in the United States from 1981 through 1997. N Engl J Med 340, 1377–1382.
Boyle, E. I., Weng, S., Gollub, J., Jin, H., Botstein, D., Cherry, J. M. & Sherlock, G. (2004). GO : : TermFinder – open source software for accessing gene ontology information and finding significantly enriched gene ontology terms associated with a list of genes. Bioinformatics 20, 3710–3715.
Cheadle, C., Vawter, M. P., Freed, W. J. & Becker, K. G. (2003). Analysis of microarray data using Z score transformation. J Mol Diagn 5, 73–81.
Chiu, S. S., Tse, C. Y. C., Lau, Y. L. & Peiris, M. (2001). Influenza A infection is an important cause of febrile seizures. Pediatrics 108, E63.
Delorme, L. & Middleton, P. J. (1979). Influenza A virus associated with acute encephalopathy. Am J Dis Child 133, 822–824.
Duchateau, P. N., Pullinger, C. R., Orellana, R. E., Kunitake, S. T., Naya-Vigne, J., O'Connor, P. M., Malloy, M. J. & Kane, J. P. (1997). Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L. J Biol Chem 272, 25576–25582.
Geiss, G. K., Salvatore, M., Tumpey, T. M. & 8 other authors (2002). Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza. Proc Natl Acad Sci U S A 99, 10736–10741.
Hayden, F. G., Fritz, R., Lobo, M. C., Alvord, W., Strober, W. & Straus, S. E. (1998). Local and systemic cytokine responses during experimental human influenza A virus infection. Relation to symptom formation and host defense. J Clin Invest 101, 643–649.[Medline]
Holzinger, A., Mayerhofer, P., Berger, J., Lichtner, P., Kammerer, S. & Roscher, A. A. (1999). Full length cDNA cloning, promoter sequence, and genomic organization of the human adrenoleukodystrophy related (ALDR) gene functionally redundant to the gene responsible for X-linked adrenoleukodystrophy. Biochem Biophys Res Commun 258, 436–442.[CrossRef][Medline]
Hong, J. J., Loiselle, C. R., Yoon, D. Y., Lee, O., Becker, K. G. & Singer, H. S. (2004). Microarray analysis in Tourette syndrome postmortem putamen. J Neurol Sci 225, 57–64.[CrossRef][Medline]
Hosack, D. A., Dennis, G., Jr, Sherman, B. T., Lane, H. C. & Lempicki, R. A. (2003). Identifying biological themes within lists of genes with EASE. Genome Biol 4, R70.[CrossRef][Medline]
Ito, Y., Ichiyama, T., Kimura, H., Shibata, M., Ishiwada, N., Kuroki, H., Furukawa, S. & Morishima, T. (1999). Detection of influenza virus RNA by reverse transcription-PCR and proinflammatory cytokines in influenza-virus-associated encephalopathy. J Med Virol 58, 420–425.[CrossRef][Medline]
Kaiser, L., Fritz, R. S., Straus, S. E., Gubareva, L. & Hayden, F. G. (2001). Symptom pathogenesis during acute influenza: interleukin-6 and other cytokine responses. J Med Virol 64, 262–268.[CrossRef][Medline]
Kasai, T., Togashi, T. & Morishima, T. (2000). Encephalopathy associated with influenza epidemics. Lancet 355, 1558–1559.[Medline]
Kash, J. C., Basler, C. F., Garcia-Sastre, A. & 7 other authors (2004). Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol 78, 9499–9511.
Kawada, J., Kimura, H., Hara, S., Ito, Y., Kawashima, H., Okuno, T. & Morishima, T. (2003a). Absence of associations between influenza-associated encephalopathy and human herpesvirus 6 or human herpesvirus 7. Pediatr Infect Dis J 22, 115–119.[CrossRef][Medline]
Kawada, J., Kimura, H., Ito, Y., Hara, S., Iriyama, M., Yoshikawa, T. & Morishima, T. (2003b). Systemic cytokine responses in patients with influenza-associated encephalopathy. J Infect Dis 188, 690–698.[CrossRef][Medline]
Kawada, J., Kimura, H. & Morishima, T. (2004). Influenza virus and febrile convulsions. J Infect Dis 189, 564–565.[Medline]
Kim, Y. H., Lee, J. H., Lim, D. S., Shim, W. J., Ro, Y. M., Park, G. H., Becker, K. G., Cho-Chung, Y. S. & Kim, M.-K. (2003). Gene expression profiling of oxidative stress on atrial fibrillation in humans. Exp Mol Med 35, 336–349.[Medline]
Law, A. J., Shannon Weickert, C., Hyde, T. M., Kleinman, J. E. & Harrison, P. J. (2004). Neuregulin-1 (NRG-1) mRNA and protein in the adult human brain. Neuroscience 127, 125–136.[CrossRef][Medline]
Morishima, T., Togashi, T., Yokota, S., Okuno, Y., Miyazaki, C., Tashiro, M. & Okabe, N. (2002). Encephalitis and encephalopathy associated with an influenza epidemic in Japan. Clin Infect Dis 35, 512–517.[CrossRef][Medline]
Moser, H. W. (2000). Disorders of very long chain fatty acids. In Nelson Textbook of Pediatrics, 16th edn, pp. 381–387. Edited by R. E. Behrman, R. M. Kliegman & H. B. Jenson. Philadelphia: W. B. Saunders.
Newland, J. G., Romero, J. R., Varman, M., Drake, C., Holst, A., Safranek, T. & Subbarao, K. (2003). Encephalitis associated with influenza B virus infection in 2 children and a review of the literature. Clin Infect Dis 36, e87–e95.[CrossRef][Medline]
Ogburn, P. L., Jr, Sharp, H., Lloyd-Still, J. D., Johnson, S. B. & Holman, R. T. (1982). Abnormal polyunsaturated fatty acid patterns of serum lipids in Reye's syndrome. Proc Natl Acad Sci U S A 79, 908–911.
Ronni, T., Sareneva, T., Pirhonen, J. & Julkunen, I. (1995). Activation of IFN-alpha, IFN-gamma, MxA, and IFN regulatory factor 1 genes in influenza A virus-infected human peripheral blood mononuclear cells. J Immunol 154, 2764–2774.[Abstract]
Skoner, D. P., Gentile, D. A., Patel, A. & Doyle, W. J. (1999). Evidence for cytokine mediation of disease expression in adults experimentally infected with influenza A virus. J Infect Dis 180, 10–14.[CrossRef][Medline]
Starko, K. M., Ray, C. G., Dominguez, L. B., Stromberg, W. L. & Woodall, D. F. (1980). Reye's syndrome and salicylate use. Pediatrics 66, 859–864.
Straussberg, R., Amir, J., Harel, L., Punsky, I. & Bessler, H. (2001). Pro- and anti-inflammatory cytokines in children with febrile convulsions. Pediatr Neurol 24, 49–53.[CrossRef][Medline]
Tong, H. H., Long, J. P., Li, D. & DeMaria, T. F. (2004). Alteration of gene expression in human middle ear epithelial cells induced by influenza A virus and its implication for the pathogenesis of otitis media. Microb Pathog 37, 193–204.[CrossRef][Medline]
Virta, M., Hurme, M. & Helminen, M. (2002). Increased plasma levels of pro- and anti-inflammatory cytokines in patients with febrile seizures. Epilepsia 43, 920–923.[CrossRef][Medline]
Wright, P. F. & Webster, R. G. (2001). Orthomyxoviruses. In Fields Virology, 4th edn, vol. 1, pp. 1533–1579. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
Received 9 November 2005;
accepted 15 February 2006.
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