Highly pathogenic H5N1 influenza viruses continue to cause concern, even though currently circulating strains are not efficiently transmitted among humans. For efficient transmission, amino acid changes in viral proteins may be required. Here, we examined the amino acids at positions 627 and 701 of the PB2 protein. A direct analysis of the viral RNAs of H5N1 viruses in patients revealed that these amino acids contribute to efficient virus propagation in the human upper respiratory tract. Viruses grown in culture or eggs did not always reflect those in patients. These results emphasize the importance of the direct analysis of original specimens.
Given the continued circulation of highly pathogenic H5N1 avian influenza viruses and their sporadic transmission to humans, the threat of a pandemic persists. However, for H5N1 influenza viruses to be efficiently transmitted among humans, amino acid substitutions in the avian viral proteins may be necessary.
Two positions in the PB2 protein affect the growth of influenza viruses in mammalian cells (3, 11, 18): the amino acid at position 627 (PB2-627), which in most human influenza viruses is lysine (PB2-627Lys) and most avian viruses is glutamic acid (PB2-627Glu), and the amino acid at position 701. PB2-627Lys is associated with the efficient replication (16) and high virulence (5) of H5N1 viruses in mice. Moreover, an H7N7 avian virus isolated from a fatal human case of pneumonia possessed PB2-627Lys, whereas isolates from a nonfatal human case of conjunctivitis and from chickens during the same outbreak possessed PB2-627Glu (2).
The amino acid at position 701 in PB2 is important for the high pathogenicity of H5N1 viruses in mice (11). Most avian influenza viruses possess aspartic acid at this position (PB2-701Asp); however, A/duck/Guangxi/35/2001 (H5N1), which is highly virulent in mice (11), possesses asparagine at this position (PB2-701Asn). PB2-701Asn is also found in equine (4) and swine (15) viruses, as well as some H5N1 human isolates (7, 9). Thus, both amino acids appear to be markers for the adaptation of H5N1 viruses in humans (1, 3, 17).
Massin et al. (13) reported that the amino acid at PB2-627 affects viral RNA replication in cultured cells at low temperatures. Recently, we demonstrated that viruses, including those of the H5N1 subtype, with PB2-627Lys (human type) grow better at low temperatures in cultured cells than those with PB2-627Glu (avian type) (6). This association between the PB2 amino acid and temperature-dependent growth correlates with the body temperatures of hosts; the human upper respiratory tract is at a lower temperature (around 33°C) than the lower respiratory tract (around 37°C) and the avian intestine, where avian influenza viruses usually replicate (around 41°C). The ability to replicate at low temperatures may be crucial for viral spread among humans via sneezing and coughing by being able to grow in the upper respiratory organs. Therefore, the Glu-to-Lys mutation in PB2-627 is an important step for H5N1 viruses to develop pandemic potential.
However, there is no direct evidence that the substitutions of PB2-627Glu with PB2-627Lys and PB2-701Asp with PB2-701Asn occur during the replication of H5N1 avian influenza viruses in human respiratory organs. Therefore, here, we directly analyzed the nucleotide sequences of viral genes from several original specimens collected from patients infected with H5N1 viruses.
Mixed PB2 population in original specimens.
To investigate the significance of PB2-627Lys and PB2-701Asn in H5N1 viruses for replication in humans, we extracted viral RNAs from six original specimens (once per specimen) using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. RNAs were reverse transcribed with SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) and an oligonucleotide complementary to the 12-nucleotide sequence at the 3′ end of the viral RNA and amplified by PCR with Pwo DNA polymerase (Roche, Basel, Switzerland) and primers specific for the PB2 segment of the H5N1 influenza virus (sequences available upon request). The PCR products were gel purified with a QIAquick gel extraction kit (Qiagen, Hilden, Germany) and cloned into pCR-Blunt II-TOPO (Invitrogen) by TOPO cloning technology (Invitrogen) and transformed into TOP10 cells (Invitrogen). Escherichia coli clones were picked, and the nucleotide sequences of the viral genes that cover the regions that include PB2-627 and -701 were analyzed. All experiments with infectious H5N1 viruses were performed under biosafety level 3 containment.
We first examined two different specimens obtained from the same patient (HN3040), believed to be directly infected with H5N1 avian viruses. One was a pharyngeal swab collected from the upper respiratory tract; the other was a tracheal aspirate from the lower respiratory tract (Table 1). Maines et al. (12) previously isolated H5N1 viruses with PB2-627Lys (A/Vietnam/1203/04 [H5N1]) from sample HN3040II and PB2-627Glu (A/Vietnam/1204/04 [H5N1]) from HN3040I (both possess PB2-701Asp) (Table 1). A direct analysis of the viral RNAs in these two original specimens revealed avian-type (no PB2-627Lys or PB2-701Asn) and human-type (PB2-627Lys or PB2-701Asn) viruses in each specimen (Fig. 1). Mixed PB2 populations in two other specimens from patient HN3030 were also observed (Fig. 1). These results suggested that although it is possible that the viruses possessing the human-type PB2 genes were already circulating in birds, human-adapted viruses likely emerged during replication in humans.
Most of the virus in HN3040I possessed human-type PB2 (11/12 clones), compared to 7/12 clones in HN3040II (Fig. 1). In the other two specimens collected from the upper respiratory tract, HN3047III and HN3062, we found only human-type PB2 amino acids (Fig. 1). On the other hand, 47% and 20% of the specimens from the lower respiratory tract, HN3030I and HN3030II, respectively, contained avian-type PB2 (Fig. 1). These results demonstrate that the human-type PB2-627Lys and PB2-701Asn are selected during virus replication in the upper respiratory tract of humans.
Virus selection during propagation in MDCK cells and embryonated chicken eggs.
Generally, MDCK cells are used for virus isolation from mammals (except horses ), and embryonated chicken eggs are used for virus isolation from birds. Human viruses with hemagglutinin mutations are selected during propagation in eggs (10, 14). Thus, these procedures may misrepresent virus populations in humans. To test this possibility, we inoculated specimens (Table 1) into MDCK cells and chicken eggs, incubated them at 37°C for 2 to 5 days, and compared the PB2 gene in the propagated viruses with the original specimens (Fig. 1). We found no viruses with avian-type PB2 in any of the specimens, indicating that under these conditions, H5N1 viruses with avian-type PB2 do not propagate in MDCK cells or chicken eggs.
To reveal the factor(s) that caused this discordance between the original specimens and the viral stocks, we inoculated HN3040I and HN3040II into MDCK cells (Fig. 2) and chicken eggs (Fig. 3), incubated them at 33°C and 37°C, harvested the cultured supernatants and allantoic fluids at various time points, and analyzed the PB2 gene in the propagated viruses.
Although most of the viruses grown in MDCK cells possessed human-type PB2, all of the viruses grown in these cells with HN3040II at a 10-fold dilution at 37°C, but not at 33°C, possessed the avian-type PB2 (Fig. 2). These results show that avian-type viruses are selected at higher temperatures in MDCK cells. The inconsistent results with HN3040II-inoculated MDCK cells at 37°C (Fig. 1 and 2B) may reflect low virus titer in the original specimen. A limited number of virions containing only human-type PB2 may have been inoculated into the cells. This also explains the lack of virus with the 100-fold-diluted HN3040II (Fig. 2B, right column).
Viruses grown in eggs were more heterogeneous than those grown in MDCK cells with HN3040 samples (Fig. 2 and 3). Since the viruses with PB2-701Asn were not detected in the original specimen, the detection of such viruses at 33°C but not at 37°C in eggs suggests that PB2-701 may promote their growth under this condition.
Our direct analysis of the viral RNAs from the original patient specimens showed that PB2-627Lys and PB2-701Asn of the H5N1 avian influenza viruses were independently selected during replication in the human respiratory tract. The direct sequencing of the samples identified a mixed population with avian-type and human-type PB2, suggesting that viruses with human-type amino acids arose under selective pressures in humans. We also demonstrated that a PB2 gene population grown in either cell culture or eggs does not always reflect that in patients. A direct analysis of the original specimens is required for accurate information on the viral genome and to monitor for prepandemic viruses.
HN3040I and HN3040II were collected from the same patient, and HN3030I and HN3030II were collected from the same patient. ID, identification.
These viruses were previously isolated by another group (12) and us (19).
We thank Susan Watson for editing the manuscript.
This work was supported by ERATO (Japan Science and Technology Agency); by a grant-in-aid for specially promoted research; by a contract research fund for the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases from the Ministry of Education, Culture, Sports, Science, and Technology; by grants-in-aid from the Ministry of Health, Labor, and Welfare of Japan; and by National Institute of Allergy and Infectious Disease Public Health Service research grants.
Finkelstein, D. B., S. Mukatira, P. K. Mehta, J. C. Obenauer, X. Su, R. G. Webster, and C. W. Naeve. 2007. Persistent host markers in pandemic and H5N1 influenza viruses. J. Virol.81:10292-10299.
Fouchier, R. A., P. M. Schneeberger, F. W. Rozendaal, J. M. Broekman, S. A. Kemink, V. Munster, T. Kuiken, G. F. Rimmelzwaan, M. Schutten, G. J. Van Doornum, G. Koch, A. Bosman, M. Koopmans, and A. D. Osterhaus. 2004. Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. Proc. Natl. Acad. Sci. USA101:1356-1361.
Gorman, O. T., R. O. Donis, Y. Kawaoka, and R. G. Webster. 1990. Evolution of influenza A virus PB2 genes: implications for evolution of the ribonucleoprotein complex and origin of human influenza A virus. J. Virol.64:4893-4902.
Hatta, M., Y. Hatta, J. H. Kim, S. Watanabe, K. Shinya, T. Nguyen, P. S. Lien, Q. M. Le, and Y. Kawaoka. 2007. Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice. PLoS Pathog.3:1374-1379.
Hiromoto, Y., T. Saito, S. Lindstrom, and K. Nerome. 2000. Characterization of low virulent strains of highly pathogenic A/Hong Kong/156/97 (H5N1) virus in mice after passage in embryonated hens' eggs. Virology272:429-437.
Ilobi, C. P., R. Henfrey, J. S. Robertson, J. A. Mumford, B. J. Erasmus, and J. M. Wood. 1994. Antigenic and molecular characterization of host cell-mediated variants of equine H3N8 influenza viruses. J. Gen. Virol.75:669-673.
Li, Z., H. Chen, P. Jiao, G. Deng, G. Tian, Y. Li, E. Hoffmann, R. G. Webster, Y. Matsuoka, and K. Yu. 2005. Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model. J. Virol.79:12058-12064.
Maines, T. R., X. H. Lu, S. M. Erb, L. Edwards, J. Guarner, P. W. Greer, D. C. Nguyen, K. J. Szretter, L. M. Chen, P. Thawatsupha, M. Chittaganpitch, S. Waicharoen, D. T. Nguyen, T. Nguyen, H. H. Nguyen, J. H. Kim, L. T. Hoang, C. Kang, L. S. Phuong, W. Lim, S. Zaki, R. O. Donis, N. J. Cox, J. M. Katz, and T. M. Tumpey. 2005. Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals. J. Virol.79:11788-11800.
Shinya, K., S. Hamm, M. Hatta, H. Ito, T. Ito, and Y. Kawaoka. 2004. PB2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of Hong Kong H5N1 influenza A viruses in mice. Virology320:258-266.
Yamada, S., Y. Suzuki, T. Suzuki, M. Q. Le, C. A. Nidom, Y. Sakai-Tagawa, Y. Muramoto, M. Ito, M. Kiso, T. Horimoto, K. Shinya, T. Sawada, T. Usui, T. Murata, Y. Lin, A. Hay, L. F. Haire, D. J. Stevens, R. J. Russell, S. J. Gamblin, J. J. Skehel, and Y. Kawaoka. 2006. Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors. Nature444:378-382.
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