| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Institut für Virologie und Antivirale Therapie, Universitätsklinikum Jena, Hans Knöll Str. 2, D-07745 Jena, Germany
2 Friedrich Loeffler Institute, Federal Research Institute for Animal Health, Institute of Molecular Biology, Südufer 10, D-17493 Greifswald - Insel Riems, Germany
3 IDT Biologika GmbH, Bereich Forschung und Entwicklung, Am Pharmapark, D-06861 Dessau-Rosslau, Germany
Correspondence
Roland Zell
Roland.Zell{at}med.uni-jena.de
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Supplementary material is available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Brown et al., 1997). In 1979, novel porcine influenza A viruses (swFLUAV) of the H1N1 subtype emerged in Europe (Pensaert et al., 1981). These viruses have eight genome segments originating from avian strains and are designated ‘avian-like’ accordingly. After the initial bird-to-swine transmission, the European avian-like swFLUAVs underwent subsequent reassortments with human influenza viruses to yield ‘human-like’ swFLUAV H3N2 (emergence 1984) and H1N2 subtypes (emergence 1994). Those subtypes have human haemagglutinin (HA) and neuraminidase (NA) segments, whereas the internal segments are avian-like (Brown, 2000). H3N2 and H1N2 reassortants spread region-wide in European swine populations and replaced the classical swine viruses. European swFLUAVs constitute distinct genetic lineages (Brown et al., 1998) and have the propensity to infect humans (Rimmelzwaan et al., 2001; Gregory et al., 2001, 2003; Claas et al., 1994). Recently, the emergence of amantadine-resistant European swFLUAVs and an alarming increase of amantadine-resistant human isolates was reported (Bright et al., 2005; Deyde et al., 2007; Marozin et al., 2002; Schmidtke et al., 2006, 2008). Amantadine, an adamantane derivative, is an antiviral compound effective against FLUAV. Despite certain side-effects and a rapid induction of resistant strains, amantadine is licensed for the prophylaxis and therapy of influenza in various countries (for review, see Hayden, 2006). It inhibits the function of the FLUAV M2 proton channel and single amino acid substitutions at positions 26, 27, 30, 31 and 34 of the M2 protein confer resistance against it (Hay et al., 1985; Abed et al., 2005). Recently, structure determination of tetrameric M2 transmembrane helices in presence and absence of amantadine and rimantadine, respectively, provided evidence for refined models describing both proton gating and viral inhibition by adamantanes (Schnell & Chou, 2008; Stouffer et al., 2008). In this study, a molecular epidemiological investigation of European swFLUAVs, analysis of their amantadine resistance phenotype and a genetic analysis of the M segment was performed. In addition, recombinant viruses which correspond to reassortant human viruses with avian-like M segments of European swFLUAVs were rescued and analysed for their amantadine susceptibility. |
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Zell et al., 2008). Additional virus isolates were obtained from collections of the Bundesinstitut für Risikoforschung (Berlin, Germany), the Niedersächsisches Landesgesundheitsamt (Hannover, Germany) and the Department of Virology, Parasitology, Immunology of Ghent University (Ghent, Belgium).
Virus RNA preparation and sequencing.
For sequencing of the viral genome, total RNA was prepared from virus-infected MDCK cells using the RNeasy Mini and QIAshredder kits (Qiagen). Reverse transcription was conducted with a primer specific to the 3'-end of genomic RNA, 20 U reverse transcriptase (Fermentas) and 5 µg RNA in a final reaction volume of 20 µl. Specific oligonucleotide primers (Schmidtke et al., 2006) and cDNA were used for the amplification of DNA employing Pfu polymerase (Promega). Amplification products were subjected to agarose gel electrophoresis and extracted employing a QIAquick Gel Extraction kit (Qiagen). Purified amplification products were sequenced by cycle sequencing using the CEQ DTCS Quick Start kit (Beckman Coulter) and analysed on a CEQ8000 sequencer (Beckman Coulter). The nucleotide sequences were submitted to GenBank (accession nos EU478795
[GenBank]
–EU478849
[GenBank]
).
Phylogenetic and molecular genetic analyses.
Nucleotide sequences were aligned manually with the help of MEGA version 4 (Tamura et al., 2007). For tree inference, Bayesian Metropolis-coupled Markov chain (MCMC) analysis was conducted with MrBayes 3.1 (Ronquist & Huelsenbeck, 2003). Four chains were calculated until convergence was reached (substitution model: GTR+G+I, four gamma categories, chain length: 1 900 000 generations). The optimal substitution model was selected in a hierarchical likelihood ratio test on the basis of the Akaike information criterion (AIC) using MODELTEST (Posada & Crandall, 1998). The tree was visualized with TreeView 1.6.6 (Page, 1996). Divergence times within the European swFLUAVs were inferred using Bayesian MCMC analysis and a relaxed clock model as implemented in BEAST 1.4.7 (Drummond & Rambaut, 2007). The dataset was analysed using the GTR+G+I substitution model, four gamma categories and a chain length of 50 000 000 generations. Two partitions into codon positions (SRD06 model, default unlink parameters) and a relaxed clock (uncorrelated lognormal) were assumed. Log parameters were sampled every 1000 generations. A maximum credibility tree was constructed with FigTree 1.1.2 (Drummond & Rambaut, 2007). Posterior probabilities of nodes, node ages (node heights) and branch lengths were displayed. Natural selection acting on each amino acid site was inferred using codon-based maximum-likelihood methods (the single likelihood ancestor counting method, SLAC; the fixed effects likelihood method, FEL; and the internal fixed effects likelihood method, IFEL) provided by the Datamonkey web server (http://www.datamonkey.org; Kosakovsky Pond & Frost, 2005). Positive selection (non-synonymous substitution rate greater synonymous substitution rate, dN>dS) and negative selection (dN<dS) was estimated as dN–dS differences. In order to compare the results of different methods, normalized dN–dS values [(dN–dS)/(codon tree length)] are presented. Co-evolution of amino acid sites was analysed with the Spidermonkey/BGM tool (Poon et al., 2007) of the Datamonkey web server. This software tool performs a Bayesian graphical model (BGM) analysis on aligned nucleotide sequences. Genotype determination was conducted with the FluGenome web server (http://www.FluGenome.org; Lu et al., 2007).
Antiviral testing.
The plaque reduction assay was used for antiviral testing. It was conducted as previously described (Schmidtke et al., 2006). Briefly, MDCK cells were infected and overlaid with 0.4 % agar supplemented with 0.001–10 µg amantadine ml–1. At day 3–4, the plaque number was determined and the IC50 was calculated. Rescued viruses were examined for amantadine susceptibility by virus titration in the presence of various amantadine concentrations according to Masuda et al. (2000). Amantadine resistance was defined as an at least 50-fold increase of IC50.
Reverse genetics.
For the generation of recombinant viruses, the M segments of two H1N1 viruses, A/sw/Potsdam/15/81 (amantadine-susceptible) and A/sw/Belzig/2/01 (amantadine-resistant), were amplified. PCR fragments were purified and cloned into pHW2000 (Hoffmann et al., 2000). The pHW plasmid system allows the rescue of recombinant A/WSN/33 (H1N1). Mixed cultures of 293T/MDCK cells were transfected with mixtures of eight plasmids (total 2 µg DNA) using FuGENE (Roche). Plasmids pHW18-1 to -8 served as positive control. Viable recombinant virus (either reassortant virus or control virus A/WSN/33) was rescued and passaged ten times in MDCK cells to ascertain viability. Reassortant virus, A/WSN/33 and both parental (non-recombinant) viruses were assayed for amantadine resistance phenotype.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Those viruses belonged either to the avian-like H1N1 or human-like H3N2 and H1N2 subtypes. In order to undertake a more comprehensive analysis, we studied additional swFLUAV strains of all three subtypes that presently circulate in Europe. The isolates were collected in Germany between 1981 and 2006. In addition, one English and three Belgian isolates were included. Because five substitutions of the M2 open reading frame strongly correlate with the amantadine-resistant phenotype (Abed et al., 2005), genotyping, i.e. sequencing of the M segment, was previously used for the analysis of amantadine resistance of human and avian influenza virus isolates (Bright et al., 2005; Deyde et al., 2007; Hurt et al., 2007). Fifty-five M sequences determined in this study as well as additional 49 (almost) complete and eight partial M sequences retrieved from GenBank were compiled to yield an alignment of M sequences of European swFLUAVs (n=112, Supplementary Table S1). This alignment included sequences of eight swFLUAV isolates from Hong Kong as well as one German turkey isolate and two zoonotic human isolates from Hong Kong and Switzerland (Gregory et al., 2001, 2003), since those viruses were found to belong to the genetic lineage of prevalent European swFLUAVs. Sequence comparison revealed that all swFLUAV isolates since 1989 have the characteristic S31N substitution of the M2 protein conferring amantadine resistance (Supplementary Table S1). In addition, double mutants with the S31N and either of the L26F, V27A or V27T substitutions were also observed in some resistant strains. The second substitution also confers amantadine resistance (Hay et al., 1985; Abed et al., 2005). Interestingly, 10 strains have the novel L26I-V27A-S31N triple substitution, another strain has L26I-V27T-S31N substitutions. Yet unknown is the significance of L26I and V27I exchanges. Moreover, a coincident R77Q substitution was observed in all resistant swFLUAV strains. A GenBank survey revealed that these S31N/R77Q exchanges were not found in any other M sequence of human or animal virus isolates (data not shown).
Antiviral testing
In previous studies amantadine resistance was deduced from genetic data and verified for some (arbitrarily) selected strains by further antiviral testing (e.g. Bright et al., 2005; Deyde et al., 2007; Hurt et al., 2007). In analogy, we confirmed the amantadine resistance phenotype of a representative fraction of our collection (29/70 randomly selected isolates=41 %) using the plaque reduction assay as described (Schmidtke et al., 2006). As expected, amantadine resistance correlates with the S31N substitution (Supplementary Table S1): all susceptible strains have a serine codon at position 31, whereas the resistant strains have the S31N substitution. Antiviral analysis confirmed the observation that strains isolated after 1989 are amantadine-resistant, whereas the elder strains are amantadine-susceptible (Table 1).
|
). These sequences served as outgroups for the phylogenetic analysis and represent genetic lineages found in (i) pigs (genotype A: classical swine), (ii) humans (genotype B), (iii) gulls (genotype C), (iv) horses (genotype D), (v) American birds (genotype E), (vi) Eurasian/Australian birds (genotype F) and (vii) a few isolates from Australian stints represent genotype G. In addition, one sequence (A/shorebird/Delaware/168/2006, H16N3) of a yet unclassified genotype was also included. Beside these sequences, the dataset comprised 105 available sequences (55 sequences determined in this study and 50 sequences of avian-like isolates retrieved from GenBank). A phylogenetic tree was inferred using Bayesian MCMC analysis. The resulting tree (Fig. 1) indicates that the M sequences of the European swFLUAVs constitute a monophyletic branch within the F genotype. This clade also includes sequences of one avian strain (A/turkey/Germany/3/91, H1N1), eight porcine H3N2 strains from Hong Kong (isolated 1999–2002) and one human isolate (A/Hong Kong/1774/99, H3N2). Additional phylogenetic analyses verified that all published sequences of these viruses clustered with European swFLUAVs (data not shown). Another avian-like isolate, A/sw/Hong Kong/168/1993 (H1N1), is clearly distinct from the European lineage of swFLUAVs, but clusters within the F genotype. Surprisingly, all amantadine-resistant swFLUAV strains (as deduced from the S31N substitution) cluster in a single clade with A/sw/Schwerin/103/89 (H1N1) being closest to the root. The respective node is supported with high posterior probability (1.0). Monophyly of the European swFLUAV lineage was verified using another alignment comprising sequences of the European swFLUAVs and additional 20 arbitrarily selected avian sequences of the F genotype (data not shown). Beside the S31N mutation, additional substitutions (A26F, L26I, V27A, V27I, A27T) could be traced. None of them emerged independently of the S31N substitution.
|
were derived from a relaxed clock analysis and reveal that the swFLUAV lineage appeared in 1979 [node age: 26.83 years before 2006, 95 % highest posterior density (HPD) interval: 26.14–33.39 years before 2006) and amantadine-resistance (S31N substitution) may have emerged in 1988 (node age: 17.8 years before 2006, range of the 95 % HPD interval: 17.09–22.55 years before 2006). 1979 as the year of emergence of European swFLUAVs was also inferred by Ludwig et al. (1995).
|
Natural selection of the M2 open reading frame
Codon-based tests were performed to identify natural selection at single amino acid sites. Positive (diversifying) selection, i.e. excess of non-synonymous substitutions, and negative (purifying) selection, i.e. excess of synonymous substitutions, were investigated using different maximum-likelihood methods (SLAC, FEL and IFEL). Several codons were suggested to be restrained by negative selection: (i) codons 30, 31 and 34 which confer amantadine resistance and (ii) codons 32, 50, 51 58, 67 and 71 (Supplementary Table S2, available in JGV Online). Positive selection was suggested for codon 19 in the IFEL method. The corresponding P-values were <0.05, indicating that less than 5 % of neutrally evolving sites may be incorrectly classified as selected. All three methods showed a similar pattern of dN–dS differences. However, the SLAC and IFEL methods were found to be more conservative, resulting in fewer suggestions for natural selection (Fig. 3, left panel). In addition, 46 of 97 codons (47.4 %) were invariant. The mean substitution rate of this dataset was 0.54 substitutions/site and the mean dN/dS was 0.60. For comparison, a dataset comprising 80 unique M2 sequences of human H1N1 and H3N2 influenza virus isolates was also included in this analysis (Supplementary Table S2, Fig. 3, right panel). The mean substitution rate of this dataset was slightly greater (0.67 substitutions/site), but the mean dN/dS value was smaller (0.44) indicating some more synonymous substitutions. For this dataset, positive selection of codons 28, 36, 54, 89 and 93 and negative selection of codons 2, 25, 29, 30, 32, 34, 35, 45, 51, 53, 58, 59, 65, 69, 71, 75 and 96 was suggested; 33 % of the codons were invariant. Negative selection of codons 30, 32, 34, 51, 58 and 71 was suggested in both datasets, but the overall patterns of the dN–dS values along the open reading frames differ at codon 2, 11–28 and 54 (Fig. 3), indicating diverse selective forces which act on the M2 proteins of porcine and human influenza A viruses.
|
, the M segment of human and porcine influenza viruses belong to different genotypes. Therefore, the question arose whether a reassortant A/WSN/33 virus with a M segment of genotype F rather than genotype B is viable. As a proof of principle, the compatibility of the avian-like M segment, the transfer of the amantadine resistance phenotype and the viability of such a potential reassortant virus was demonstrated. For the generation of recombinant viruses, the M segments of an amantadine-susceptible strain (A/sw/Potsdam/15/81, H1N1) and an amantadine-resistant strain (A/sw/Belzig/2/01, H1N1) were cloned into pHW2000 (Hoffmann et al., 2000) and two recombinant A/WSN/33 viruses (H1N1) were rescued using the eight plasmid system previously described (Hoffmann et al., 2000). These viruses were designated A/WSN/33xA/sw/Potsdam/15/81-M (amantadine-susceptible) and A/WSN/33xA/sw/Belzig/2/01-M (amantadine-resistant). Subsequently, the rescued viruses were tested for viability in serial passages and the amantadine susceptibility was assayed by determination of the mean dose–response curves. They demonstrate that, independently of the genomic background, the M segment of A/sw/Potsdam/15/81 confers amantadine sensitivity (IC50 <0.25 µg ml–1), whereas the M segment of A/sw/Belzig/2/01 leads to amantadine resistance (IC50>10 µg ml–1) (Fig. 4).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The significance of the L26I substitution, which is also found in amantadine-resistant H5N1 isolates from Vietnam, Thailand, Cambodia and Malaysia of 2004–2005 (Li et al., 2004; Hurt et al., 2007) is still unclear. Two further observations are noteworthy: (i) the emergence of amantadine resistance of swFLUAVs was a single event as indicated by the monophyly of the resistance-conferring M segment; (ii) subsequently, amantadine-susceptible H1N1 viruses were displaced by resistant strains. As concluded from the phylogenetic tree, the resistance-conferring M segment was then transferred to H3N2 viruses by reassortment. The earliest resistant H3N2 strain was isolated in 1992. Then, in 1994, the first H1N2 strains emerged in the UK; those strains resulted from reassortment (Brown et al., 1998) and are amantadine-resistant. Due to the few sequences of that time available in GenBank, the exact date of the S31N/R77Q substitutions and the space of time needed for the successive replacement of the amantadine-susceptible strains remain obscure, but it is very likely that this time span might have been short. The molecular clock analysis indicates that the S31N substitution might have occurred in 1988 (95 % HPD: 1983–1989). In fact, no sequenced European swFLUAV isolated since 1989 is amantadine-susceptible, whereas none of the viruses of 1979–1987 is resistant (Fig. 1, Supplementary Table S1). Since 92 amantadine-resistant strains were collected by different laboratories throughout Europe over a period of 17 years, biased sampling that favoured isolation of resistant strains is highly unlikely. The sequences of the present study are predominantly from German isolates, but Belgian, British, Dutch, French, Italian, Spanish, Swiss and Hong Kong strains were also included in order to compile the most comprehensive collection of M sequences currently available.
Spontaneous emergence of amantadine-resistant porcine influenza viruses in the absence of the drug is a rare event. Worldwide, only three independent incidents have been documented. Beside the emergence in Europe, only two clades were found in an analysis of available sequence data (approx. 180 sequences) of classical swine isolates (H1N1) and reassortant H1N2, H3N2 and H3N1 strains derived thereof (data not shown). As there are no indications that pigs ever have been treated with amantadine in Europe, the emergence of amantadine resistance has very likely a natural cause. The reason for the astounding spread of resistant porcine virus strains remains elusive, but may not be unusual. In recent years, the prevalence of adamantane-resistant human influenza viruses has been increasing rapidly (compare Bright et al., 2005; Deyde et al., 2007). Previous investigations indicate that two mechanisms may account for the amantadine resistance of human FLUAVs: spontaneous substitutions occurring with low incidence (Ziegler et al., 1999), and the selection of resistant variants upon antiviral therapy (Shiraishi et al., 2003). Interestingly, excessive use of adamantane inhibitors may not explain upsurge of resistant human strains in many countries. Simonsen et al. (2007), observed that a 4+4 segment reassortment event favoured resistant strains due to fitness-enhancing mutations at other genomic sites. For swFLUAVs, the sequence data do not allow the demonstration of such a hitchhiking effect (data not shown). Also, selection of resistant strains upon antiviral therapy appears to be an unlikely mechanism, but a bottleneck effect might be a reasonable explanation (Domingo & Holland, 1997).
Our codon-based tests for natural selection revealed that codons 30, 31 and 34, which are known to confer amantadine resistance, are likely to be negatively selected. For codon 31, it appears that only four states are allowed: either AGT/C (encoding serine; amantadine-susceptible) or AAT/C (encoding asparagine; amantadine-resistant). The remaining four serine codons (UCA, UCG, UCC, UCT), though coding for the wild-type amino acid, were not observed – neither in porcine nor in human influenza viruses. One may argue that the S31N substitution is the only viable mutation at this site which maintains both structural constraints (i.e. formation of a proper transmembrane helix which is able to tetramerize) and functional constraints of the M2 protein (i.e. proton channel activity even in the presence of amantadine). Substitutions of alanine 30 and glycine 34 were not observed in the swFLUAVs (Supplementary Table S1).
A concerning issue is the possibility that the swFLUAV M segment might be introduced into human influenza viruses by reassortment. In order to substantiate the biological significance of this hypothetical threat, we demonstrate the viability of A/WSN/33 reassortants with M segments of European swFLUAVs. Accordingly, the amantadine susceptibility of the reassortant was altered (Fig. 4), indicating amantadine resistance in vivo. Whereas the M segment of the European swFLUAVs belongs to genotype F, all human subtypes have a M segment of the B genotype (Fig. 1). The viability of our rescued H1N1 reassortant along with its amantadine resistance suggests a possible scenario in which the M segments of human viruses could be replaced by an avian-like M segment in the field. Beside circulating amantadine resistant human H3N2 and H1N1 viruses, human–swine reassortants with an avian-like M segment could be an additional, however yet unforeseeable menace. A similar concern was also expressed by Claas et al. (1994) and Gregory et al. (2001). Therefore, both intense surveillance and elucidation of the acting molecular selection mechanisms are indispensable.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Astrahan, P., Kass, I., Cooper, M. A. & Arkin, I. T. (2004). A novel method of resistance for influenza against a channel-blocking antiviral drug. Proteins 55, 251–257.[CrossRef][Medline]
Bright, R. A., Medina, M. J., Xu, X., Perez-Oronoz, G., Wallis, T. R., Davis, X. R., Pavinelli, L., Cox, N. J. & Klimov, A. I. (2005). Incidence of adamantane resistance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern. Lancet 366, 1175–1181.[CrossRef][Medline]
Brown, I. H. (2000). The epidemiology and evolution of influenza viruses in pigs. Vet Microbiol 74, 29–46.[CrossRef][Medline]
Brown, I. H., Ludwig, S., Olsen, C. W., Hannoun, C., Scholtissek, C., Hinshaw, V. S., Harris, P. A., McCauley, J. W., Strong, I. & Alexander, D. J. (1997). Antigenic and genetic analyses of H1N1 influenza A viruses from European pigs. J Gen Virol 78, 553–562.[Abstract]
Brown, I. H., Harris, P. A., McCauley, J. W. & Alexander, D. J. (1998). Multiple genetic reassortment of avian and human influenza A viruses in European pigs, resulting in the emergence of an H1N2 virus of novel genotype. J Gen Virol 79, 2947–2955.[Abstract]
Claas, E. C. J., Kawaoka, Y., De Jong, J. C., Masurel, N. & Webster, R. G. (1994). Infection of children with avian-human reassortant influenza virus from pigs in Europe. Virology 204, 453–457.[CrossRef][Medline]
Deyde, V. M., Xu, X., Bright, R. A., Shaw, M., Smith, C. B., Zhang, Y., Shu, Y., Gubarewa, L. V., Cox, N. J. & Kimov, A. I. (2007). Surveillance of resistance to adamantanes among influenza A(H3N2) and A(H1N1) viruses isolated worldwide. J Infect Dis 196, 249–257.[CrossRef][Medline]
Domingo, E. & Holland, J. J. (1997). RNA virus mutations and fitness for survival. Annu Rev Microbiol 51, 151–178.[CrossRef][Medline]
Drummond, A. J. & Rambaut, A. (2007). BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol 7, 214[CrossRef][Medline]
Gregory, V., Lim, W., Cameron, K., Bennett, M., Klimov, A., Hall, H., Cox, N., Hay, A. J. & Lin, Y. (2001). Infection of a child in Hong Kong by an influenza A H3N2 virus closely related to viruses circulating in European pigs. J Gen Virol 82, 1397–1406.
Gregory, V., Bennett, M., Thomas, Y., Kaiser, L., Wunderli, W., Matter, H., Hay, A. & Lin, Y. P. (2003). Human infection by a swine influenza A (H1N1) virus in Switzerland. Arch Virol 148, 793–802.[CrossRef][Medline]
Hay, A. J., Wolstenholme, A. J., Skehel, J. J. & Smith, M. H. (1985). The molecular basis of the specific anti-influenza action of amantadine. EMBO J 4, 3021–3024.[Medline]
Hayden, F. G. (2006). Antivirals for influenza: historical perspectives and lessons learned. Antiviral Res 71, 372–378.[CrossRef][Medline]
Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G. & Webster, R. G. (2000). A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A 97, 6108–6113.
Hurt, A. C., Selleck, P., Komadina, N., Shaw, R., Brown, L. & Barr, I. G. (2007). Susceptibility of highly pathogenic A(H5N1) avian influenza viruses to the neuraminidase inhibitors and adamantanes. Antiviral Res 73, 228–231.[CrossRef][Medline]
Kosakovsky Pond, S. L. & Frost, S. D. W. (2005). Datamonkey: rapid detection of selective pressure on individual sites of codon alignments. Bioinformatics 21, 2531–2533.
Li, K. S., Guan, Y., Wang, J., Smith, G. J., Xu, K. M., Duan, L., Rahardjo, A. P., Puthavathana, P., Buranathai, C. & other authors (2004). Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 430, 209–213.[CrossRef][Medline]
Lu, G., Rowley, T., Garten, R. & Donis, R. O. (2007). FluGenome: a web tool for genotyping influenza A virus. Nucleic Acids Res 35, W275–W279.
Ludwig, S., Stitz, L., Planz, O., Van, H., Fitch, W. M. & Scholtissek, S. (1995). European swine virus as a possible source for the next influenza pandemic? Virology 212, 555–561.[CrossRef][Medline]
Marozin, S., Gregory, V., Cameron, K., Bennett, M., Valette, M., Aymard, M., Foni, E., Barigazzi, G., Lin, Y. & Hay, A. (2002). Antigenic and genetic diversity among swine influenza A H1N1 and H1N2 viruses in Europe. J Gen Virol 83, 735–745.
Masuda, H., Suzuki, H., Oshitani, H., Saito, R., Kawasaki, S., Nishikawa, M. & Satoh, H. (2000). Incidence of amantadine-resistant influenza A viruses in sentinel surveillance sites and nursing homes in Niigata, Japan. Microbiol Immunol 44, 833–839.[Medline]
Nardelli, L., Pascucci, S., Gualandi, G. L. & Loda, P. (1978). Outbreaks of classical swine influenza in Italy in 1976. Zentralbl Veterinarmed B 25, 853–857.[Medline]
Page, R. D. M. (1996). TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358.
Pensaert, M., Ottis, K., Vandeputte, J., Kaplan, M. M. & Bachmann, P. A. (1981). Evidence for the natural transmission of influenza A virus from wild ducks to swine and its potential importance for man. Bull World Health Organ 59, 75–78.[Medline]
Poon, A. F. Y., Lewis, F. I., Kosakovsky Pond, S. L. & Frost, S. D. W. (2007). An evolutionary-network model reveals stratified interactions in the V3 loop of the HIV-1 envelope. PLoS Comput Biol 3, e231[CrossRef][Medline]
Posada, D. & Crandall, K. A. (1998). MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817–818.
Rimmelzwaan, G. F., de Jong, J. C., Bestebroer, T. M., van Loon, A. M., Claas, E. C. J., Fouchier, R. A. M. & Osterhaus, A. D. M. E. (2001). Antigenic and genetic characterization of swine influenza A (H1N1) viruses isolated from pneumonia patients in the Netherlands. Virology 282, 301–308.[CrossRef][Medline]
Ronquist, F. & Huelsenbeck, J. P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574.
Schmidtke, M., Zell, R., Bauer, K., Krumbholz, A., Schrader, C., Süss, J. & Wutzler, P. (2006). Amantadine resistance among porcine H1N1, H1N2, and H3N2 influenza A viruses isolated in Germany between 1981 and 2001. Intervirology 49, 286–293.[CrossRef][Medline]
Schmidtke, M., Bauer, K., Ludwig, N. & Wutzler, P. (2008). Emergence and phylogenetic relationships of amantadine-resistant human H3N2 influenza A viruses in Germany in the season 2005/2006. Int J Antimicrob Agents 32, 192–198.[CrossRef][Medline]
Schnell, J. R. & Chou, J. J. (2008). Structure and mechanism of the M2 proton channel of influenza A virus. Nature 451, 591–595.[CrossRef][Medline]
Shiraishi, K., Mitamura, K., Sakai-Tagawa, Y., Goto, H., Sugaya, N. & Kawaoka, Y. (2003). High frequency of resistant viruses harboring different mutations in amantadine-treated children with influenza. J Infect Dis 188, 57–61.[CrossRef][Medline]
Simonsen, L., Viboud, C., Grenfell, B. T., Dushoff, J., Jennings, L., Smit, M., Macken, C., Hata, M., Gog, J. & other authors (2007). The genesis and spread of reassortment human influenza A/H3N2 viruses conferring adamantane resistance. Mol Biol Evol 24, 1811–1820.
Stouffer, A. L., Acharya, R., Salom, D., Levine, A. S., Di Constanzo, L., Soto, C. S., Tereshko, V., Nanda, V., Stayrock, S. & DeGrado, W. F. (2008). Structural basis for the function and inhibition of an influenza virus proton channel. Nature 451, 596–599.[CrossRef][Medline]
Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596–1599.
Zell, R., Motzke, S., Krumbholz, A., Wutzler, P., Herwig, V. & Dürrwald, R. (2008). Novel reassortant of swine influenza H1N2 virus in Germany. J Gen Virol 89, 271–276.
Ziegler, T., Hemphill, M. L., Ziegler, M. L., Perez-Oronoz, G., Klimov, A. I., Hampson, A. W., Regnery, H. L. & Cox, N. J. (1999). Low incidence of rimantadine resistance in field isolates of influenza A viruses. J Infect Dis 180, 935–939.[CrossRef][Medline]
Received 11 September 2008;
accepted 30 November 2008.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
