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1 Department of Pathobiological Sciences, University of Wisconsin–Madison, WI 53706, USA
2 Graduate School of Medicine, Tohoku University, Sendai 980-8575, Japan
3 Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
4 Avian Zoonosis Research Center, Tottori University, Tottori 680-8553, Japan
5 International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
6 CREST, Japan Science and Technology Agency, Saitama 332-0012, Japan
Correspondence
Yoshihiro Kawaoka
kawaoka{at}ims.u-tokyo.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Avian Zoonosis Research Center, Tottori University, Tottori 680-8553, Japan. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). During replication, the viral genomes evolve and adapt to their new hosts. Recently, an equine influenza virus was transmitted to dogs and caused lethal infections (Crawford et al., 2005). Despite few differences between the amino acid sequences of the canine and equine isolates, the molecular mechanism by which this equine influenza virus adapted to its canine host remains unknown.
Genomic changes in influenza A viruses during adaptation in a new host have been studied in mice. When Brown et al. (2001) adapted a human influenza A virus in mice, the adapted virus was more pathogenic and had undergone 11 amino acid changes; however, further experiments to determine specific amino acid substitutions that were critical for mouse adaptation were not done. Another mouse adaptation study with chicken-adapted seal-derived H7N7 virus demonstrated polymerase activity-enhancing mutations in the mouse-adapted virulent mutant (Gabriel et al., 2005). These mutations mapped to amino acids at positions 615 in PA (PA-615), 701 and 714 in PB2 (PB2-701 and PB2-714) and 319 in NP (NP-319).
The H5N1 influenza A viruses isolated from humans in the 1997 Hong Kong outbreak have been divided into two groups based on their virulence in mice (Gao et al., 1999; Katz et al., 2000), which generally correlates with their virulence in humans (Katz et al., 2000). The Glu-to-Lys substitution at PB2-627 (PB2-E627K) was identified as a mutation that is responsible for the difference in virulence of some of these viruses (Hatta et al., 2001). Moreover, the PB2-E627K substitution was identified following a single passage of avian H5N1 viruses in mice (Lipatov et al., 2003; Mase et al., 2005), further suggesting its relevance in mouse adaptation. The importance of the PB2-E627K substitution for virus replication in humans was substantiated following the isolation of H5N1 viruses with this mutation from humans in more recent outbreaks (Smith et al., 2006). The same mutation was found in a virus that caused a fatal infection in a human, but not among viruses from individuals who developed only conjunctivitis or among chickens during an H7N7 virus outbreak (Fouchier et al., 2004). Recently isolated H5N1 viruses from ducks have been shown to carry a PB2-D701N mutation that also plays an important role in increased virulence in mice (Li et al., 2005).
The above studies show that influenza A viruses with different genetic backgrounds can acquire different mutations during adaptation in mice. This knowledge led us to examine the adaptation of an equine influenza A virus in mice. It has been shown previously that A/equine/London/1416/73 (Eq/Lon; H7N7) is lethal in mice without adaptation; however, after nine passages in this animal, it acquires enhanced virulence, including neuropathogenicity (Eq/Lon-MA; Kawaoka, 1991). Eq/Lon causes pneumotropic lethal infection with rare brain invasion in mice; however, Eq/Lon-MA replicates in systemic organs, including brain. To understand the molecular mechanism of the enhanced virulence of this equine influenza virus, reverse genetics were used to make mutant viruses possessing specific amino acid alterations in order to identify the critical mutations that occur during mouse adaptation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Viruses were grown in 11-day-old embryonated chicken eggs and infectivity titres of virus in the allantoic fluid were determined by calculating the 50 % egg infectious dose (EID50). Virus fluid was stored at –80 °C until use.
Cells.
Madin–Darby canine kidney (MDCK) cells were grown in minimum essential medium with Eagle salts containing 5 % newborn calf serum, 4 mM L-glutamine and antibiotics. Human 293T embryonic kidney cells, a derivative of 293 cells that express the simian virus 40 T antigen gene constitutively, were maintained in Dulbecco's modified Eagle's medium (glucose concentration, 4.5 g l–1) supplemented with 10 % fetal bovine serum (FBS), 4 mM L-glutamine and antibiotics. LA-4 mouse lung adenoma cells (ATCC CCL-196) were maintained in F12K nutrient mixture (Kaighn's modification) supplemented with 15 % FBS, 2 mM L-glutamine and antibiotics. WEHI-3B mouse myelomonocytic leukaemia cells were maintained in RPMI 1640 medium with 10 % FBS and antibiotics.
Molecular cloning and sequencing of the viral genes.
Viral RNA (vRNA) of Eq/Lon and Eq/Lon-MA was extracted from virus-containing allantoic fluid by using an RNA extraction kit (RNeasy; Qiagen). The viral genes were amplified by the RT-PCR method by using two pairs of gene-specific oligonucleotide primers that possessed the BsmBI site (the sequences of the oligonucleotide primers can be supplied upon request), SuperScript III (Invitrogen) and PfuUltra (Stratagene). Amplified RT-PCR products were purified by using a gel extraction kit (Qiagen). To sequence the Eq/Lon-MA strain, the products were cloned into pT7 Blue vector (Perfectly Blunt kit; Novagen) or Zero Blunt TOPO vector (Invitrogen). At least three clones per gene were sequenced. For the Eq/Lon strain, the RT-PCR products were sequenced directly after gel purification.
Construction of plasmids.
The cloned gene segments of the Eq/Lon-MA strain were digested with BsmBI and then subcloned into the BsmBI site of pHH21, which contains cloned influenza viral cDNA under the control of the human RNA polymerase I promoter, as well as the mouse RNA polymerase I terminator (referred to as PolI plasmids; Neumann et al., 1999). Consequently, transfection of these plasmids into 293T cells resulted in the synthesis of influenza vRNAs. To generate mutant strains, PCR-based site-directed mutagenesis with primer pairs containing point mutations was used.
Reverse genetics.
To generate the Eq/Lon-MA strain and its mutant counterparts from cloned cDNA, reverse genetics were performed by using eight PolI plasmids that encoded each Eq/Lon-MA gene, or the Eq/Lon-MA gene with various mutations, plus four plasmids that encoded the WSN (A/WSN/33) PA, PB1, PB2 and NP, each of which was cloned into pCAGGS/MCS under the control of the chicken 
-actin promoter. The PolI plasmids and the protein expression plasmids were transfected into 293T cells by using Trans-IT LT-1 (Panvera). The supernatant of the 293T culture was harvested at 48 h after transfection and inoculated into 11-day-old embryonated chicken eggs. After harvesting the allantoic fluid, the EID50 was determined. The virus fluid was stored at –80 °C until use. The Eq/Lon-MA strain generated by reverse genetics was designated Eq/Lon-MA-RG.
Generation of recombinant Eq/Lon-MA(VSV-G) and its mutant virus.
A recombinant virus expressing the glycoprotein of vesicular stomatitis virus (VSV-G) instead of haemagglutinin (HA) and green fluorescent protein (GFP) instead of neuraminidase (NA) was generated by transfecting 293T cells with pPolINA[183]GFP[157]Met(–) (Fujii et al., 2003), pPolIHA[9]VSVG[80] (Watanabe et al., 2003), the remaining six pPolI constructs derived from the Eq/Lon-MA strain and four plasmids expressing the WSN PA, PB1, PB2 and NP proteins [designated Eq/Lon-MA(VSV-G)]. To generate a mutant Eq/Lon-MA(VSV-G) that possessed glutamic acid at position 627 of PB2 instead of lysine (as is found in Eq/Lon-MA), a pPolI-PB2 plasmid that was mutated to introduce the Glu-to-Lys mutation at this position was used.
Infection of recombinant viruses in cell culture.
MDCK, LA-4 or WEHI-3B cells were infected with recombinant viruses at an m.o.i. of 1 and incubated at 37 °C for 24 h. At 9 and 24 h post-infection (p.i.), GFP fluorescence was observed under an inverted fluorescence microscope (Nikon). Growth rates of the recombinant viruses were determined by infecting MDCK or LA-4 cells with recombinant viruses at an m.o.i. of 10–3 and then incubating them at 37 °C. At 24, 48, 72 and 96 h p.i., supernatants were collected and titrated in MDCK cells by plaque assays.
Experimental infection of mice.
To determine the dose lethal to 50 % of infected mice (MLD50), 4-week-old female BALB/c mice were inoculated intranasally with 50 µl 10-fold serially diluted virus fluid. Virus titres in the lungs and brain of mice inoculated with Eq/Lon-MA or its PB2-627E mutant [Eq/Lon-MA-RG (PB2-627E)] were determined at days 3 and 6 p.i. Organs were weighed and homogenized in PBS and the amount of virus in the homogenates was determined by inoculating 100 µl serial dilutions of the homogenates into 11-day-old embryonated chicken eggs.
Pathology examination.
Four-week-old female BALB/c mice were inoculated intranasally with 50 µl (107 EID50) Eq/Lon-MA-RG virus. Mice were euthanized on day 6 p.i. and their lungs and brains were removed and fixed in 10 % phosphate-buffered formalin. Specimens were then dehydrated, embedded in paraffin and cut into sections 5 µm thick. For viral antigen detection, sections were processed for immunostaining by the two-step dextran polymer method (DAKO), with a rabbit polyclonal antibody to A/whistling swan/Shimane/83 (H5N3) used as the primary antibody.
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RESULTS |
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). Arginine at PB1-614 is rare, as is glycine, although glycine at 614 in PB1 has been detected in some human isolates, including an H7N3 virus in British Columbia (Hirst et al., 2004). Most equine and avian viruses have glutamic acid at position 627 in PB2, whereas most authentic human viruses possess lysine at this site. In fact, half of the human H5N1 viruses possess lysine at this position (Table 1). Interestingly, the amino acid at PB2-627 in swine viruses differs depending on the origin of the virus: avian-like swine viruses possess glutamic acid, whereas classic swine and human viruses possess lysine. Therefore, in contrast to humans and birds, the selective pressure for this residue in pigs is limited. Equine viruses and most avian viruses possess serine at position 65 in PA. Human viruses possess mainly leucine at this position and tyrosine has not been found in any human viruses. It is interesting to note that the 1918 virus possessed the avian-like serine at this position, suggesting that this amino acid may be involved in host-range adaptation; however, the selective pressure imposed on this residue does not seem to be strong (Table 1). As with PB2-627, the amino acid at this position in swine viruses differs depending on the origin of virus.
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). To determine the amino acid substitution(s) responsible for the difference in mouse virulence between the Eq/Lon and the Eq/Lon-MA strains, mutant viruses that possessed at least one of the amino acid substitutions detected in the Eq/Lon-MA strain were made. Of the four substitutions, PA-S65Y and PB1-R614G had the most limited effect on virulence, whereas PB2-E627K and PB1-K578Q had an appreciable effect, with PB2-E627K increasing virulence by 1000-fold as measured by MLD50 (Table 2). Interestingly, a mutant expressing PB2-E627K and PB1-K578Q (mutant 5) was less pathogenic than a mutant that expressed only PB2-E627K (mutant 1), even though PB1-K578Q alone (mutant 3) or in combination with PA-S65Y and PB1-R614G (mutant 7) enhanced virus virulence. The reason for this finding remains unknown.
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No appreciable difference in GFP expression was observed in MDCK cells infected with recombinant virus possessing lysine or glutamic acid at PB2-627; however, GFP expression was appreciably higher in LA-4 and WEHI-3B cells infected with recombinant virus with lysine at this position compared with glutamic acid (Fig. 1). These results indicate that the PB2-E627K substitution promotes viral protein production in mouse cells.
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). These findings indicate that PB2-E627K alone enhances replication of the equine virus in mouse cells.
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, Fig. 3). Thus, PB2-627K may enhance the ability of the virus to invade the brains of mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Here, it was found that the substitution PB2-E627K is the most important mutation for increased virulence and replication of an H7N7 equine influenza A virus in mice. The PB2-E627K substitution is known to enhance growth of avian influenza A viruses in mice and possibly in humans (Hatta et al., 2001; Katz et al., 2000; Lipatov et al., 2003; Mase et al., 2005; Naffakh et al., 2000). In fact, the importance of this mutation in host adaptation was first reported in 1993 with respect to the replication of avian/human reassortant viruses in MDCK cells (Subbarao et al., 1993). Thus, although mutations other than that at position 627 of PB2 can enhance virus replication in mice and contribute to virus adaptation to this animal, the Glu-to-Lys mutation at position 627 of PB2 appears to be one of the critical mutations in mouse adaptation of influenza A viruses with diverse genetic backgrounds.
Although it has been demonstrated that this amino acid substitution at PB2-627 enhances viral polymerase activity (Gabriel et al., 2005; Naffakh et al., 2000), the molecular mechanism by which it affects virulence remains unknown. The importance of the amino acid at PB2-627 appears to differ among mammals. In ferrets, for example, some of the H5N1 viruses with glutamic acid at this position are lethal, although the majority of the lethal H5N1 viruses possess lysine and none of the non-lethal viruses possess lysine (Katz et al., 2000; Maines et al., 2005; Salomon et al., 2006). Interestingly, H5N1 viruses isolated from tigers also possess PB2-627K, suggesting that lysine at this position provides a growth advantage to the virus in this animal. Several investigators have shown that, during replication of the H5N1 viruses in mice, the Glu-to-Lys mutation at PB2-627 can occur (Lipatov et al., 2003; Mase et al., 2005), further supporting the importance of this mutation in mice. Most authentic human influenza A viruses possess PB2-627K, including more than half of the H5N1 viruses isolated from humans. However, most avian viruses possess PB2-627E, with the notable exception of the H5N1 viruses isolated during an outbreak among wild waterfowl in Qinghai Lake, China, in 2005 (Chen et al., 2005; Liu et al., 2005) and the viral descendants of those responsible for the outbreak (Chen et al., 2006). Moreover, an H7N7 virus possessing the PB2-627K substitution was isolated from a lethal human case, but not from a non-lethal one in which the primary symptom was conjunctivitis (Fouchier et al., 2004). These findings indicate that similar constraints act upon PB2 during virus replication in both mice and humans. Therefore, the mouse model may contribute to our understanding of the pathogenic mechanisms related to this amino acid substitution during human infection.
Some amino acids at specific positions in PB1, PB2, PA and NP (i.e. the proteins responsible for RNA transcription and replication) are characteristic of the human or avian viruses (Katz et al., 2000). Some of these amino acids are introduced randomly and maintained, whereas others are selected during adaptation of the virus in humans. Of four amino acid substitutions that occurred during Eq/Lon adaptation in mice, PA-S65Y and PB2-E627K occurred at positions where avian and human viruses differ, although at PA-65, most human viruses possess leucine, not tyrosine as is found in Eq/Lon-MA. Whereas introduction of the PB2-E627K and PB1-K578Q substitutions alone enhanced virulence of Eq/Lon, the other two mutations exhibited an additive effect on virulence enhancement. How these amino acid substitutions lead to increased virus replication and virulence in mice remains unknown, but elucidating the mechanism by which they alter virus growth in a new host will boost our understanding of interspecies transmission of influenza A viruses and may help to prevent future pandemics.
A recent human case suggests that the H5N1 virus invades the central nervous system (de Jong et al., 2005). As with mice, H5N1 viruses have been shown to invade the brain of ferrets. Interestingly, PB2-627K does not appear to be essential for this property (Govorkova et al., 2005; Maines et al., 2005; Rowe et al., 2003; Zitzow et al., 2002). However, our findings here suggest that PB2-627K may enhance neuroinvasiveness in mice (Table 3
). Thus, whether the PB2-E627K substitution is required for neurotropism in humans should be examined.
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ACKNOWLEDGEMENTS |
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Received 23 July 2006;
accepted 19 October 2006.
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