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Short Communication |
Department of Infectious Diseases, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan
Correspondence
Masaji Mase
masema{at}affrc.go.jp
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ABSTRACT |
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, 2005; Li et al., 2004; Liu et al., 2005; Mase et al., 2005b). Incidents of the causative H5N1 virus being transmitted directly from birds to humans occurred in 1997 and 2003 in Hong Kong (Peiris et al., 2004; Subbarao et al., 1998; Yuen et al., 1998). From 2004 to 2006, these viruses were transmitted to humans in Azerbaijan, Cambodia, China, Egypt, Indonesia, Iraq, Thailand, Turkey and Vietnam, and this transmission resulted in over 100 deaths (WHO, 2006). Therefore, an epidemic of H5N1 avian influenza still poses a serious threat to public health.
Mice have been shown to be a good mammalian model for the human H1N1, H2N2 and H3N2 influenza viruses. Therefore, mice were used for the H5N1 influenza viruses as well, and it was revealed that recent isolates of the H5N1 influenza virus replicated well in mice without prior adaptation (Gao et al., 1999; Guan et al., 2002, 2004; Lipatov et al., 2003; Lu et al., 1999; Nishimura et al., 2000; Shortridge et al., 1998).
In Japan, an outbreak of highly pathogenic H5N1 avian influenza in chickens was confirmed in 2004 for the first time (Mase et al., 2005b). The causative H5N1 viruses in Japan were genetically close to A/chicken/Shantou/4231/2003, termed genotype V, which belongs to a genotype different from that of the dominant epidemic viruses in South-East Asian countries, such as Indonesia, Thailand and Vietnam (i.e. genotype Z) (Li et al., 2004). This suggested that multiple H5N1 virus genotypes have been circulating and are associated with the occurrence of the serious outbreaks in poultry in Asian countries.
The H5N1 virus isolated in Japan during the 2003–2004 outbreaks was able to replicate in mice without prior adaptation, but the the isolate was less virulent than the Hong Kong 1997 H5N1 viruses isolated from humans (Gao et al., 1999; Mase et al., 2005b). However, it was previously revealed experimentally that highly pathogenic H5N1 variants could be selected rapidly in mice after a single passage (Lipatov et al., 2003). Here, we describe the biological and pathological characterization of recovered viruses with rapidly increasing virulence to mice by only one amino acid substitution in the complete genome after a single passage in mice.
The Japanese H5N1 virus A/chicken/Yamaguchi/7/2004 (Ck/Yama/7/04), isolated from chickens (Mase et al., 2005b), was used in this study. To prepare the original virus stock for this study, virus was propagated once in the allantoic cavity of embryonated eggs at 37 °C for 1–2 days and then stored at –80 °C until use. All experiments using the live H5N1 virus were performed in biosafety level 3 facilities under the recommended procedures.
First, we examined the pathogenicity to mice of the original Ck/Yama/7/04 strain. Six-week-old female BALB/c mice (n=18; SLC Japan) were used in all experiments. The mice were anaesthetized by pentobarbital injection and 50 µl infectious virus [106 50 % egg infectious dose (EID50)] diluted in PBS was inoculated intranasally (i.n.). The mice were checked daily for clinical signs and mortality for 14 days post-infection (p.i.). Reisolation of the virus from the brain, lung, liver, spleen and kidney of the dead mice was conducted immediately after the animals' deaths, as described previously (Mase et al., 2005a).
Fourteen mice in total died during the observation period, and the virus was reisolated from the brains of all dead mice. The mean time to death of the mice infected with the original Ck/Yama/7/04 strain was 8.3 days. To examine the extent of mutations, we determined the nucleotide sequences of isolates from the mouse brains. First, partial nucleotide sequences of the PB2 gene, which was related to the virulence to mice of the Hong Kong H5N1/97 viruses, of all isolates from the mouse brains were determined. Reverse transcription, PCR amplification and sequencing were performed as described previously (Mase et al., 2005b).
All viruses recovered from the brains of dead mice inoculated with the Ck/Yama/7/04 strain had an amino acid substitution from glutamic acid (Glu) to lysine (Lys) at position 627 of the PB2 protein, as shown by partial nucleotide sequencing of the PB2 gene. Next, the complete nucleotide sequences of all segments of five chosen isolates (termed mouse brain variants; MBVs) recovered from five respective mice were determined as described previously (Mase et al., 2005a). Interestingly, as for the results of the complete nucleotide sequencing of all segments of the five chosen recovered isolates, four viruses have only one amino acid substitution at position 627 of the PB2 protein from Glu to Lys. From these viruses, one isolate was selected and designated mouse brain variant A (MBV-A). The remaining isolate has an additional amino acid substitution, methionine (Met) to isoleucine (Ile), at position 531 of the haemagglutinin (HA), and was designated MBV-B. In the following tests, the MBV-A and -B strains were used as mouse variants derived from the original Ck/Yama/7/04 strain.
Next, the pathogenicity to mice of the original virus and MBVs were compared. Six-week-old female BALB/c mice (SLC Japan) were used. Fifty per cent mouse infectious dose (MID50) and 50 % mouse lethal dose (MLD50) titres were determined by inoculating groups of eight mice i.n. with serial 10-fold dilutions of virus according to the method described by Lu et al. (1999). Three days later, four mice from each group were euthanized and the lungs were collected and homogenized. The homogenates were frozen at –80 °C and later thawed for ease of handling. Solid debris was pelleted by centrifugation and the supernatants were titrated for virus infectivity in eggs. The four remaining mice in each group were checked daily for disease signs and death for 14 days p.i. The MID50 and MLD50 titres were calculated by the method of Reed & Muench (1938).
The scores of MID50 and MLD50 of the two MBVs were 5 EID50 and 8.9 EID50, respectively. However, the scores of MID50 and MLD50 of the original Ck/Yama/7/04 strain were 5x103 EID50 and 5x105 EID50, respectively. Comparing these scores, the MLD50 of two MBVs were increased by 5x104 times over that of the original Ck/Yama/7/04 strain.
Next, a histopathological examination of the mice infected with the original and MBV-A viruses was performed. Six-week-old female BALB/c mice (n=18 for each of the original and MBV-A viruses) were inoculated i.n. with 50 µl virus (106 EID50). A total of three sacrificed or dead mice were examined on day 3 and another three on day 6 p.i. The major organs were removed and fixed in 10 % neutral phosphate-buffered formalin, then processed routinely and stained with haematoxylin and eosin (HE) for histopathological examination. A section mounted on silane-coated slide glass was heated by microwave as described previously (Tanimura et al., 2004) and goat anti-influenza A virus polyclonal antibody (Chemicon) was applied. The primary antibody was allowed to incubate for 30 min at room temperature and then detected by the application of horseradish peroxidase anti-goat Ig conjugate (Histofine Simple Stain; Nichirei Inc.). Diaminobenzidine (Nichirei Inc.) served as the substrate chromogen and haematoxylin was used as a counterstain. For the negative control, the primary antibody was applied to tissues of a normal mouse. In addition, the primary antibody was substituted with normal goat serum (DakoCytomation). There was no non-specific staining in these negative controls, except for the mast cells in the connective tissue, which showed endogenous peroxidase activity in their cytoplasmic granules. The viral titres of the brain, lung, liver, spleen and kidney of these mice were determined as described previously (Mase et al., 2005b).
The results of the histopathological and immunohistochemical analyses are summarized in Table 1. In the respiratory organs, such as the lungs and nasal turbinates, severe damage and viral antigens were detected earlier in the MBV-A-infected mice than in those infected with the original strain (Table 1b; Fig. 1a–d). Viral antigen-positive alveolar cells appeared to be alveolar macrophages or type II alveolar epithelial cells. In the central nervous system (CNS), whereas viral antigens were detected in the MBV-A-infected mice at day 3 p.i., they were not detected in the original Ck/Yama/7/04-infected mice until day 6 p.i. (Table 1b; Fig. 1e). Lesions and viral antigens were only observed in the olfactory bulbs of the MBV-A strain-infected mice (Fig. 1f).
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). The virus titres of the variant virus-infected mice were higher than those of the original virus-infected mice. At day 6 p.i., viruses were recovered from all examined organs except the kidneys collected from MBV-A strain-infected mice.
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). The molecular basis of the lethal virulence of the recent epidemic H5N1 viruses to humans in these countries is not understood completely, but in the case of Hong Kong H5N1 in 1997, the molecular basis of the high virulence was studied in the mouse model (Hatta et al., 2001). By using reverse genetics, a Lys at residue 627 in the PB2 protein and polybasic cleavage site in HA were found to be crucial for the highly virulent and systematic replication of the A/Hong Kong/483/97 (H5N1) virus in mice. In particular, the presence of Lys leads to more aggressive virus replication, overwhelming the host's defence mechanisms and resulting in high mortality rates in mice (Gabriel et al., 2005; Massin et al., 2001; Naffakh et al., 2000; Shinya et al., 2004; Subbarao et al., 1993). Interestingly, the Dutch H7N7 virus isolated in 2003 from humans had Lys at position 627 of the PB2 protein, whereas H7N7 in 2003 isolated from chickens maintained Glu at this position (Fouchier et al., 2004). The PB2s of most human H1N1, H2N2 and H3N2 influenza viruses examined thus far possess Lys at position 627, whereas those of their progenitor avian viruses examined thus far all contain Glu at this position (Wright & Webster, 2001). Taken together, it was suggested that the substitution of Glu to Lys at this position arises easily in transmission from birds to mammals.
Here, we demonstrated experimentally that the Japanese H5N1 viruses isolated in 2004 substituted their amino acid at position 627 of the PB2 protein from Glu to Lys after a single passage in mice, with increasing virulence. A variant virus containing Lys at position 627 replicated more rapidly than the original virus containing Glu at this position. This substitution may be a first step for adaptation to mammals, as human H1N1, H2N2 and H3N2 influenza viruses have Lys at position 627 of the PB2 protein (Wright & Webster, 2001). Comparing the MID50 and MLD50 of both the MBV-A and MBV-B viruses, the amino acid substitution at position 531 of the HA seemed not to be critical for high virulence. By pathological analysis of the MBV-A-infected mice, severe pneumonia was observed in the earlier phase (3 days p.i.) and encephalomyelitis was also observed at the later phase (6 days p.i.). Viruses were recovered from the internal organs of the MBV-A-infected mice, but the virus titres of the tissues at 6 days p.i. were lower than those at 3 days p.i., except in the brain. Moreover, histopathological analysis suggested that the viruses isolated from the kidney might be derived from adipose tissues around this organ. However, the appearance of mild lesions and the detection of viral antigens in the olfactory bulb in MBV-A-infected mice suggested rapid replication and spreading in this tissue. The routes of invasion of the HK483 strain into the CNS were suggested previously to be through afferent fibres of the olfactory, vagal, trigeminal and sympathetic nerves following replication in the respiratory mucosa (Park et al., 2002). The invasion of the MBV-A strain into the CNS in mice seemed to be facilitated strongly by these three routes compared with the original strain.
Although the Japanese H5N1 viruses were different in H5N1 genotype (genotype V) from the South-East Asian isolates of H5N1, including those from Thailand and Vietnam (genotype Z), the PB2 protein of Japanese H5N1 (Ck/Yama/7/04) was very similar (about 99.5 %) to that of Thailand H5N1 (A/goose/Thailand/79/2004) at the amino acid level. In fact, many recent H5N1 isolates (genotype Z) from mammals have this substitution at position 627 of the PB2 protein (Govorkova et al., 2005; Keawcharoen et al., 2004; Puthavathana et al., 2005). Urgent measures to deal with a possible pandemic, such as the development and application of effective vaccines and the stockpiling of anti-influenza drugs, are needed.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Chen, H., Smith, G. J. D., Zhang, S. Y., Qin, K., Wang, J., Li, K. S., Webster, R. G., Peiris, J. S. M. & Guan, Y. (2005). Avian flu: H5N1 virus outbreak in migratory waterfowl. Nature 436, 191–192.[CrossRef][Medline]
Fouchier, R. A. M., Schneeberger, P. M., Rozendaal, F. W. & 11 other authors (2004). Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. Proc Natl Acad Sci U S A 101, 1356–1361.
Gabriel, G., Dauber, B., Wolff, T., Planz, O., Klenk, H.-D. & Stech, J. (2005). The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc Natl Acad Sci U S A 102, 18590–18595.
Gao, P., Watanabe, S., Ito, T., Goto, H., Wells, K., McGregor, M., Cooley, A. J. & Kawaoka, Y. (1999). Biological heterogeneity, including systemic replication in mice, of H5N1 influenza A virus isolates from humans in Hong Kong. J Virol 73, 3184–3189.
Govorkova, E. A., Rehg, J. E., Krauss, S. & 11 other authors (2005). Lethality to ferrets of H5N1 influenza viruses isolated from humans and poultry in 2004. J Virol 79, 2191–2198.
Guan, Y., Peiris, J. S. M., Lipatov, A. S., Ellis, T. M., Dyrting, K. C., Krauss, S., Zhang, L. J., Webster, R. G. & Shortridge, K. F. (2002). Emergence of multiple genotypes of H5N1 avian influenza viruses in Hong Kong SAR. Proc Natl Acad Sci U S A 99, 8950–8955.
Guan, Y., Poon, L. L. M., Cheung, C. Y. & 10 other authors (2004). H5N1 influenza: a protean pandemic threat. Proc Natl Acad Sci U S A 101, 8156–8161.
Hatta, M., Gao, P., Halfmann, P. & Kawaoka, Y. (2001). Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science 293, 1840–1842.
Keawcharoen, J., Oraveerakul, K., Kuiken, T. & 12 other authors (2004). Avian influenza H5N1 in tigers and leopards. Emerg Infect Dis 10, 2189–2191.[Medline]
Li, K. S., Guan, Y., Wang, J. & 19 other authors (2004). Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 430, 209–213.[CrossRef][Medline]
Lipatov, A. S., Krauss, S., Guan, Y., Peiris, M., Rehg, J. E., Perez, D. R. & Webster, R. G. (2003). Neurovirulence in mice of H5N1 influenza virus genotypes isolated from Hong Kong poultry in 2001. J Virol 77, 3816–3823.
Liu, J., Xiao, H., Lei, F. & 11 other authors (2005). Highly pathogenic H5N1 influenza virus infection in migratory birds. Science 309, 1206.
Lu, X., Tumpey, T. M., Morken, T., Zaki, S. R., Cox, N. J. & Katz, J. M. (1999). A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans. J Virol 73, 5903–5911.
Mase, M., Eto, M., Tanimura, N., Imai, K., Tsukamoto, K., Horimoto, T., Kawaoka, Y. & Yamaguchi, S. (2005a). Isolation of a genotypically unique H5N1 influenza virus from duck meat imported into Japan from China. Virology 339, 101–109.[CrossRef][Medline]
Mase, M., Tsukamoto, K., Imada, T. & 11 other authors (2005b). Characterization of H5N1 influenza A viruses isolated during the 2003–2004 influenza outbreaks in Japan. Virology 332, 167–176.[CrossRef][Medline]
Massin, P., van der Werf, S. & Naffakh, N. (2001). Residue 627 of PB2 is a determinant of cold sensitivity in RNA replication of avian influenza viruses. J Virol 75, 5398–5404.
Naffakh, N., Massin, P., Escriou, N., Crescenzo-Chaigne, B. & van der Werf, S. (2000). Genetic analysis of the compatibility between polymerase proteins from human and avian strains of influenza A viruses. J Gen Virol 81, 1283–1291.
Nishimura, H., Itamura, S., Iwasaki, T., Kurata, T. & Tashiro, M. (2000). Characterization of human influenza A (H5N1) virus infection in mice: neuro-, pneumo- and adipotropic infection. J Gen Virol 81, 2503–2510.
Park, C. H., Ishinaka, M., Takada, A., Kida, H., Kimura, T., Ochiai, K. & Umemura, T. (2002). The invasion routes of neurovirulent A/Hong Kong/483/97 (H5N1) influenza virus into the central nervous system after respiratory infection in mice. Arch Virol 147, 1425–1436.[CrossRef][Medline]
Peiris, J. S. M., Yu, W. C., Leung, C. W. & 9 other authors (2004). Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet 363, 617–619.[CrossRef][Medline]
Puthavathana, P., Auewarakul, P., Charoenying, P. C. & 7 other authors (2005). Molecular characterization of the complete genome of human influenza H5N1 virus isolates from Thailand. J Gen Virol 86, 423–433.
Reed, L. J. & Muench, H. (1938). A simple method of estimating fifty percent endpoints. Am J Hyg 27, 493–497.
Shinya, K., Hamm, S., Hatta, M., Ito, H., Ito, T. & Kawaoka, Y. (2004). PB2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of Hong Kong H5N1 influenza A viruses in mice. Virology 320, 258–266.[CrossRef][Medline]
Shortridge, K. F., Zhou, N. N., Guan, Y. & 12 other authors (1998). Characterization of avian H5N1 influenza viruses from poultry in Hong Kong. Virology 252, 331–342.[CrossRef][Medline]
Subbarao, E. K., Kawaoka, Y. & Murphy, B. R. (1993). Rescue of an influenza A virus wild-type PB2 gene and a mutant derivative bearing a site-specific temperature-sensitive and attenuating mutation. J Virol 67, 7223–7228.
Subbarao, K., Klimov, A., Katz, J. & 13 other authors (1998). Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science 279, 393–396.
Tanimura, N., Imada, T., Kashiwazaki, Y., Shamusudin, S., Syed Hassan, S., Jamaluddin, A., Russell, G. & White, J. (2004). Reactivity of anti-Nipah virus monoclonal antibodies to formalin-fixed, paraffin-embedded lung tissues from experimental Nipah and Hendra virus infections. J Vet Med Sci 66, 1263–1266.[CrossRef][Medline]
WHO (2006). Cumulative number of confirmed human cases of avian influenza A/(H5N1) reported to WHO. http://www.who.int/csr/disease/avian_influenza/country/cases_table_2006_06_16/en/index.html
Wright, P. F. & Webster, R. G. (2001). Orthomyxoviruses. In Fields Virology, 4th edn, pp. 1538–1579. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
Yuen, K. Y., Chan, P. K. S., Peiris, M. & 8 other authors (1998). Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancet 351, 467–471.[CrossRef][Medline]
Received 20 January 2006;
accepted 29 July 2006.
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