Highly pathogenic avian influenza viruses (HPAIV) originate from avirulent precursors but differ from all other influenza viruses by the presence of a polybasic cleavage site in their hemagglutinins (HA) of subtype H5 or H7. In this study, we investigated the ability of a low-pathogenic avian H5N1 strain to transform into an HPAIV. Using reverse genetics, we replaced the monobasic HA cleavage site of the low-pathogenic strain A/Teal/Germany/Wv632/2005 (H5N1) (TG05) by a polybasic motif from an HPAIV (TG05poly). To elucidate the virulence potential of all viral genes of HPAIV, we generated two reassortants carrying the HA from the HPAIV A/Swan/Germany/R65/06 (H5N1) (R65) plus the remaining genes from TG05 (TG05-HAR65) or in reversed composition the mutated TG05 HA plus the R65 genes (R65-HATG05poly). In vitro, TG05poly and both reassortants were able to replicate without the addition of trypsin, which is characteristic for HPAIV. Moreover, in contrast to avirulent TG05, the variants TG05poly, TG05-HAR65, and R65-HATG05poly are pathogenic in chicken to an increasing degree. Whereas the HA cleavage site mutant TG05poly led to temporary non-lethal disease in all animals, the reassortant TG05-HAR65 caused death in 3 of 10 animals. Furthermore, the reassortant R65-HATG05poly displayed the highest lethality as 8 of 10 chickens died, resembling “natural” HPAIV strains. Taken together, acquisition of a polybasic HA cleavage site is only one necessary step for evolution of low-pathogenic H5N1 strains into HPAIV. However, these low-pathogenic strains may already have cryptic virulence potential. Moreover, besides the polybasic cleavage site, the additional virulence determinants of H5N1 HPAIV are located within the HA itself and in other viral proteins.
Citation: Bogs J, Veits J, Gohrbandt S, Hundt J, Stech O, Breithaupt A, et al. (2010) Highly Pathogenic H5N1 Influenza Viruses Carry Virulence Determinants beyond the Polybasic Hemagglutinin Cleavage Site. PLoS ONE 5(7): e11826. https://doi.org/10.1371/journal.pone.0011826
Editor: Linqi Zhang, Tsinghua University, China
Received: February 22, 2010; Accepted: July 5, 2010; Published: July 27, 2010
Copyright: © 2010 Bogs et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Forschungssofortprogramm Influenza of the German government (FSI 2.44) and European Commission [SSPE-CT-2006-44372 (Innflu)]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Highly pathogenic avian influenza viruses (HPAIV) are the causative agents of fowl plague ,  which causes devastating economic losses in gallinaceous poultry. In addition, several HPAIV strains are able to infect humans and, therefore, are considered as potential precursors for future influenza pandemics . For infection, the envelope glycoprotein hemagglutinin (HA) precursor HA0 requires proteolytic cleavage by cellular proteases into the two subunits HA1 and HA2. Mammalian and low-pathogenic avian influenza A viruses (LPAIV) carry an HA cleavage site with a monobasic motif susceptible to trypsin-like proteases which confine viral replication to the respiratory or gastrointestinal tract. In contrast, HPAIV possess a polybasic HA cleavage site cleavable by furin , , which is ubiquitous and thus supports systemic viral replication. This polybasic HA cleavage site is the prime virulence determinant of HPAIV , ,  which originate from LPAIV precursors , , , , , , , , . Acquisition of a furin recognition motif was shown to result from different events such as recombination of the HA gene with 28S ribosomal RNA  or with sequences encoding other viral proteins like the nucleoprotein (NP) gene of an unrelated virus  or the HA and matrix protein genes (M) from the same virus . An alternative proposed mechanism is polymerase slippage on template regions with stable secondary structures , . In mammalian influenza viruses, virulence determinants have been attributed to the HA , , , , , NA , , NS1 , , , , , NP and polymerase proteins , , , , , , . In HPAIV, beside the polybasic HA cleavage site, the caspase cleavage motif in the M2 protein and deletions within the NA stalk region were associated with increased virulence , , , , , . Furthermore, introduction of the NS gene from an H5N1 HPAIV into an H7N1 fowl plague strain rendered it virulent for mice . Recently, we demonstrated that the acquisition of a polybasic cleavage site by an LPAIV H3N8 strain is not sufficient for immediate transformation into an HPAIV, and that additional virulence determinants other than the polybasic HA cleavage site are required . However, it remained to be analyzed whether H5 or H7 LPAIV, which are considered HPAIV precursors, have to undergo further evolutionary changes prior to or after acquisition of a polybasic cleavage site. Therefore, we addressed in this study the question whether a polybasic cleavage site engineered into the HA of an H5N1 LPAIV leads to immediate transformation into an HPAIV. To elucidate the virulence potential of all viral genes of H5N1 HPAIV in chicken further, we generated two H5N1 reassortants carrying an HPAIV HA plus the remaining LPAIV genes, or, in reversed composition, the LPAIV HA with engineered polybasic cleavage site plus the HPAIV genes.
Generation of Recombinant Viruses
As parental strains we used a recent H5N1 LPAIV isolated in Germany in 2005, A/Teal/Germany/Wv632/2005 (H5N1)  as well as the first HPAIV H5N1 isolate derived from the outbreak in wild swans on the island of Rügen in February 2006, A/Swan/Germany/R65/06 (H5N1) . First, plasmid-based reverse genetics systems were established for both strains, resulting in the recombinant viruses TG05 (this study) and R65 , respectively. To introduce a polybasic HA cleavage site into TG05 (H5N1), we replaced its monobasic HA cleavage site with a polybasic motif from HPAIV A/Duck/Shanghai/13/01 (H5N1) by site-directed mutagenesis and used this plasmid for generation of the mutant TG05poly. Considering possible structural constraints within the HA of the parental TG05, we also adapted the amino acids adjacent to the cleavage site to those from the HA of A/Duck/Shanghai/13/01 (H5N1) (Table 1). Furthermore, we rescued two reassortants carrying the HA from HPAIV R65 plus the remaining seven genes from TG05 (TG05-HAR65), or, in reversed composition, the mutated TG05 HA plus the remaining R65 genes (R65-HATG05poly). Remarkably, we could not rescue a mutant of TG05 with the polybasic cleavage site region from R65 suggesting structural impairments of the mutated HA.
TG05poly has in-vitro properties of an HPAIV
To address the question whether the HA cleavage site mutant and the two reassortants are dependent on trypsin for multicycle replication, we performed plaque assays. Whereas the parental TG05 required exogenous trypsin for plaque formation, all the viruses with a polybasic HA cleavage site, TG05poly, TG05-HAR65, and R65-HATG05poly, yielded plaques independent of trypsin (Fig. 1). Proteolytic HA cleavage was analyzed by immunoblotting. Corresponding to plaque assays, the HA0 precursor of TG05 remained uncleaved in the absence of trypsin in contrast to HA0 of TG05poly, TG05-HAR65, and R65-HATG05poly (Fig. 2), which were cleaved into the HA1 and HA2 fragments. The HA0 cleavage was incomplete in all viruses studied. Multicycle growth kinetics of TG05 showed a strong dependence on exogenous trypsin as the virus failed to replicate efficiently in the absence of trypsin. TG05poly replicated both in the presence and in the absence of trypsin, although with a delay compared with the other viruses. However, both reassortant viruses TG05-HAR65 and R65-HATG05poly replicated to high titers irrespective of trypsin (Fig. 3). In summary, these results demonstrate that TG05poly, TG05-HAR65, and R65-HATG05poly undergo multicycle replication in the absence of trypsin in contrast to their parent virus TG05 and, thus, display HPAIV phenotypes in-vitro.
Plaque assays of TG05 in comparison with TG05poly, TG05-HAR65, and R65-HATG05poly on MDCK cells in the presence and in the absence of trypsin.
Western blots of DF-1 cell lysates infected with TG05, TG05poly, TG05-HAR65 or R65-HATG05poly at multiplicity of infection of 0.1 incubated in the presence (T) and in the absence (-) of trypsin.
Pathogenicity in chicken
To investigate the virulence of TG05, TG05poly, TG05-HAR65, and R65-HATG05poly in chicken, 10 animals each were infected with 105 pfu of the respective virus oculonasally, and observed daily for clinical symptoms for 10 days (Table 2 and Fig. 4). In contrast to the low-pathogenic parental virus TG05 which proved to be completely innocuous as expected, the HA cleavage site mutant TG05poly and the two reassortants TG05-HAR65 and R65-HATG05poly were pathogenic in chicken to an increasing degree. Infection with TG05poly led to temporary non-lethal disease in all animals (predominantly mild symptoms such as ruffled feathers, depression or diarrhea mainly from day 3 to 6). After inoculation with TG05-HAR65 3 of 10 animals died, whereas R65-HATG05poly displayed the highest lethality as 8 of 10 infected chickens died, thus exhibiting the phenotype of a natural HPAIV (Table 2).
Daily clinical score after oculonasal inoculation with 105 pfu of TG05 (blue), TG05poly (magenta), TG05-HAR65 (yellow) or R65-HATG05poly (red). The birds were observed for 10 days for clinical signs and classified as healthy (0), ill (1), severely ill (2), or dead (3); the daily clinical score was calculated from the sum of individual clinical scores from all birds divided by the number of animals per group (10 chickens).
Organ tropism and tissue lesions in chicken
On day 4 post inoculation, samples from cerebellum, cerebrum, lung, nose, trachea, caecum, duodenum, kidney, and pancreas from three (TG05, TG05poly and TG05-HAR65) and four (R65-HATG05poly) additionally infected birds per group were removed and investigated for viral spread in organs and the extent of lesions in affected tissues. No influenza virus antigen or histomorphological lesions were found in organs of TG05 infected birds. In one of the three TG05poly-inoculated birds, single positive neurons, pneumocytes II, endothelial cells, lymphocytes, and macrophages could be detected in cerebrum, lung, nasal conchae, trachea, caecum, and duodenum. Furthermore, in one TG05-HAR65-infected bird, small clusters of infected cells were present in cerebellum and cerebrum suggesting that already the introduction of the R65 HA facilitates neurotropism.
All four R65-HATG05poly-infected birds exhibited larger and more intensely stained clusters of infected cells within cerebellum, cerebrum, and nasal mucosae; several birds displayed single positive cells in lung, trachea, caecum, duodenum, kidney, and pancreas. After infection with the recombinant HPAIV R65 , the picture of infected cells appeared more intense (Table 3, Fig. 5). These results demonstrate a broader organ tropism of R65-HATG05poly-inoculated birds compared with TG05poly-inoculated birds demonstrating the relevance of the other viral genes for systemic spread.
Immunohistochemical detection of influenza A virus nucleoprotein (brown) in lung and cerebellum from chickens sacrificed on day 4 post inoculation with 105 pfu of TG05 or R65-HATG05poly, and from moribund chickens sacrificed on day 2 post inoculation with 106 TCID50 R65.
On day 10 post inoculation, organs were removed from four surviving birds each of the TG05, TG05poly and TG05-HAR65-infected groups. No antigen was detectable in tissues of these birds, except for a few positive neurons and glial cells in the cerebellum from one TG05-HAR65-infected bird, and in the cerebrum from two surviving R65-HATG05poly-infected animals, as well as inflammatory cells in the nasal mucosa from one of those birds, demonstrating the recovery of the animals (Table S1).
Taken together, the presence of influenza virus antigen corresponded to degeneration and necrosis of the affected tissues accompanied by lymphocytic and histiocytic infiltration. Extent of organ tropism and severity of lesions corroborate with the course of disease. In HPAIV, viral neurotropism can be facilitated by the HA gene solely and systemic spread is mediated also by the internal protein genes.
Influenza virus virulence is a polygenic trait which had been established by generating reassortants neurovirulent for mice from two apathogenic strains, and reassortants apathogenic in chicken from two virulent strains , . Furthermore, reassortment studies based on two H5N1 HPAIV strains revealed that the exchange of the HA, NP, M2 or NS1 proteins resulted in increased mortalities and expanded tissue tropism in chicken. Moreover, replacement of the PB2 or PB1 proteins led to decreased replication in tissues and, consequently, a decrease in virulence . These findings can be attributed to involvement of these proteins in virulence or to constraints of gene segment exchanges by reassortment in HPAIV. However, the prime determinant of virulence is the polybasic HA cleavage site , ,  as its conversion into a monobasic motif invariably resulted in loss of virulence . On the other hand, introduction of such a polybasic motif into the HA cleavage site of a low-pathogenic H3N8 strain did not lead to transformation into an HPAIV indicating the existence of additional virulence determinants in the HA and/or the other viral proteins .
To shed more light on virulence determinants required for evolution of HPAIV which, in nature, derive from low-pathogenic precursors of subtypes H5 or H7 , , , , , , , , , we addressed the question whether a low-pathogenic avian strain of the H5N1 subtype would transform into a highly pathogenic strain after introduction of a polybasic HA cleavage site. For that purpose, we replaced the monobasic HA cleavage site from TG05 by a polybasic motif from the HPAIV A/Duck/Shanghai/13/01 (H5N1) resulting in the mutant TG05poly. Remarkably, attempts to obtain a similar mutant with the HA cleavage site of HPAIV R65 were not successful indicating structural incompatibilities with the TG05 HA.
Although TG05poly was able to replicate in the absence of trypsin in cell-culture, and, thus, exhibits the in-vitro phenotype of an HPAIV, this mutant caused only temporary mild disease in chicken and, accordingly, could be detected only in isolated single cells accompanied with minor lesions in affected organs. These observations differ from those obtained by the H3N8 polybasic cleavage site mutants generated from the LPAIV A/Duck/Ukraine/1/1963 which caused considerably less symptoms in chicken  compared with TG05poly indicating that the low-pathogenic H5N1 strain already carried cryptic virulence determinants. Correspondingly, repeated air sac inoculation of a low-pathogenic H5N3 isolate in 1-day-old-chickens did not lead to high virulence for 4-to-6-week-old chickens, whereas only two further passages in brain yielded an HPAIV .
To demonstrate the existence of virulence determinants besides the polybasic cleavage site in the HA or in other genes of H5N1 HPAIV, we generated two reassortants, TG05-HAR65 carrying the HA from the HPAIV R65 plus the remaining seven genes from TG05 and, in reverse gene constellation, R65-HATG05poly composed of the mutated TG05 HA plus the other R65 genes. Whereas TG05-HAR65 already had a lethality of 30%, R65-HATG05poly caused a lethality of 80%, displaying the phenotype of a “natural” HPAIV. These findings indicate that in H5N1 HPAIV the HA gene alone provides for a certain level of virulence, whereas the complete viral genetic background influences pathogenicity to a major extent.
Compared with the very broad organ tropism of the HPAIV R65 in chicken , the mutant TG05poly and the two reassortants TG05-HAR65 and R65-HATG05poly displayed generally less viral spread in organs and less extensive lesions in surrounding tissues. However, spread and lesions increased with the two reassortants from which R65-HATG05poly exhibited the phenotype of an authentic HPAIV. This finding again emphasizes the relevance of HA and the other viral genes for virulence. Moreover, all birds which had viral antigen detectable in inner organs, i.e. those infected by TG05poly, TG05-HAR65 or R65-HATG05poly also exhibited a neuronal infection. Thus, the presence of the HPAIV R65 HA alone in a LPAIV background suffices for viral neurotropism which is considered pivotal for the fatal course of fowl plague , , .
Previous studies on HPAIV strains revealed that (1) removal of the polybasic HA cleavage site results in a drastic decrease in pathogenicity ; (2) virulence is a multigenic trait as reassortants derived from HPAI strains can be attenuated , two LPAI strains can reassort to a highly virulent virus  and reassortants with exchanged HA, NP, M or NS, PB2 or PB1 gene have an altered virulence ; (3) NA stalk deletion leads to a certain increase in virulence in an LPAIV ; and (4) NS1 can increase virulence of an HPAIV further . Most of these studies started from HPAIV and measured a decline in pathogenicity, which demonstrates that the identified alterations were required for full expression of virulence, or resulted from reassortment events which are prone to incompatibilities. No study so far addressed the question whether acquisition of a polybasic cleavage site is sufficient to transform a LPAIV H5 precursor into a highly pathogenic H5 virus. However, knowledge on this problem is most relevant, since it is required for development of a science-based assessment of the risk of HPAIV emergence from LPAIV field strains. Therefore, we introduced a polybasic HA cleavage site into the low-pathogenic H5N1 strain TG05, a potential HPAI precursor virus, in order to model the early evolution of HPAIV. This approach complements the current knowledge on the relative importance of virulence determinants and HPAIV evolution from the opposite point of view.
Taken together, acquisition of a polybasic HA cleavage site is only one necessary step for evolution of H5N1 HPAIV from low-pathogenic precursor strains, which may already have cryptic virulence potential. Moreover, besides the polybasic HA cleavage site, additional virulence determinants of HPAIV are located within the HA itself and in the other viral proteins. Knowledge of these virulence factors is crucial for an assessment of the risk of transformation of any LPAIV to HPAIV which is highly relevant for the control of notifiable LPAIV H5 or H7 infections in poultry.
Material and Methods
The animal experiments were evaluated by the responsible ethics committee of the State Office for Agriculture, Food Safety and Fishery in Mecklenburg-Western Pomerania (LALFF M-V) and gained governmental approval (registration number LALLF M-V/TSD/7221.3-1.1-018/07).
Cells and Viruses
Human embryonic kidney 293T  and Madin-Darby canine kidney (MDCK) cells  were maintained in DMEM containing 10% fetal calf serum (FCS). Chicken fibroblast cells (DF-1) were cultured in ISCOVES DMEM containing 10% FCS in the absence of antibiotics. The low-pathogenic avian influenza A virus A/Teal/Germany/Wv632/2005 (H5N1) (TG05) was isolated from a healthy green winged teal (Anas crecca) . The sequences of cloned viral genes have been submitted to Genbank (accession numbers CY061882-9). A/Swan/Germany/R65/06 (H5N1) (R65) was isolated from a dead swan during the HPAIV outbreak in Rügen, Germany, 2006 (Genbank accession numbers DQ464354-DQ464361) . Both native viruses were propagated in 11-day-old embryonated chicken eggs.
Generation of recombinant viruses
Viral genes from TG05 were cloned into the plasmid pHWSccdB as described in . Site-directed mutagenesis of the HA cleavage site region was performed by the Quikchange™ protocol (primer sequences available upon request). All recombinant viruses were rescued essentially as described  with the addition of plasmids expressing the polymerase proteins and the nucleoprotein genes from A/PR/8/34 (H1N1) (a kind gift from Peter Palese) except for R65-HATG05poly. The recombinant R65  differs from the native isolate  by 4 nucleotide exchanges in the PB2 gene (DQ464357): C1009T corresponding to the amino acid replacement L329F, in the PB1 gene (DQ464361): A1603G and G1604T corresponding to the amino acid replacement S527V, and in the HA gene (DQ464354): C1737T in non-coding region. TG05 was propagated in embryonated eggs, all other recombinant viruses were grown on MDCK cells. Gene composition and HA cleavage site of the generated viruses were verified by sequencing of amplicons from reverse transcription-PCR (data not shown). All viruses with polybasic HA cleavage site were handled under BSL3+ conditions.
Plaque assay and growth curves
Plaque assays were performed on MDCK cells as described previously  with 400 µl inoculum either in the presence of 2 µg/ml N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma, Taufkirchen, Germany), or in the absence of trypsin. For determination of growth curves, DF-1 cells were inoculated at a multiplicity of infection of 10−3 in the presence of 1 µg/ml TPCK-treated trypsin or in the absence of any exogenous protease. From two independent experiments, supernatants were harvested at 0, 8, 24, 48, and 72 h post inoculation and resulting infectious virus was titrated by plaque assay on MDCK cells in the presence of trypsin.
DF-1 cells were infected at a multiplicity of infection of 0.1 in the presence of either 1.0 µg/ml TPCK-treated trypsin or no exogenous protease in ISCOVES DMEM and 0.2% bovine serum albumin (BSA) (MP Medicals, Heidelberg) until a cytopathic effect appeared (48 h for TG05, TG05-HAR65, and R65-HATG05poly; 7 d for TG05poly). Cells were lysed with 4× Laemmli buffer  containing 4% sodium dodecyl sulfate (SDS) and were inactivated by heating at 95°C for 5 min. Lysates were separated on a 10%-SDS polyacrylamid gel and electrotransferred to a nitrocellulose membrane. For detection of HA, a polyclonal rabbit antibody to the HA protein from A/Chicken/Vietnam/P41/2005 (H5N1), expressed by a vaccinia virus vector , (1∶20,000; incubated 1 h at room temperature) and as secondary antibody a rabbit-specific goat immunoglobulin G fragment conjugated with horseradish peroxidase (1∶10,000; 1 h at room temperature, Biovision, USA) were used. Antibody binding was visualized by chemiluminescence (Supersignal West Pico Chemiluminescence Kit from Pierce, Bonn).
Ten 2-week-old White Leghorn specific-pathogen-free chickens per group were infected oculonasally with 105 PFU per animal. Each bird was observed daily for 10 days for clinical signs and classified as healthy (0), ill (1) (exhibiting one of the following: respiratory symptoms, depression, diarrhea, cyanosis, edema, or central nervous symptoms), severely ill (2) (severe or more than one of the previously mentioned symptoms), or dead (3) as described previously . When birds are too sick to eat or drink, they are killed humanely and scored as dead to the next observation day .
Histopathology and immunohistochemistry
Samples from cerebellum, cerebrum, lung, nose, trachea, caecum, duodenum, kidney, and pancreas of additionally infected chicken were taken on day 4 and 10 post infection, formalin-fixed and processed for paraffin-wax-embedding according to standardized procedures. Immunohistochemical detection of influenza A virus nucleoprotein (NP) and hematoxylin eosin staining was performed as described .
For R65 , samples from cerebellum and lung were taken on day 2 from two 3-week old chickens infected oculonasally with 106 TCID50/animal.
We thank E. Hoffmann and R. G. Webster for the pHW2000 plasmid and Peter Palese for the pCAGGS(PR8)-PB2, -PB1, -PA, and -NP expression plasmids. We are very grateful to A. Globig and T. Harder for providing us with influenza A virus A/Teal/Germany/Wv632/2005 (H5N1), to Mikhail Matrosovich for the gift of MDCK cells and to Cindy Meinke, Kathrin Müller, Anne Brandenburg, Thorsten Arnold, Georg Bauer, and Gerda Busch for very skillful technical assistance.
Conceived and designed the experiments: JV OS TM JS. Performed the experiments: JB JV SG JH AB. Analyzed the data: JB JV AB JT TM JS. Contributed reagents/materials/analysis tools: JB SG JH OS JS. Wrote the paper: JB AB JT TM JS.
- 1. Schaefer W (1955) Vergleichende seroimmunologische Untersuchungen über die Viren der Influenza und der klassischen Geflügelpest. Z Naturforsch 10b: 81–91.
- 2. Swayne DE (2007) Understanding the complex pathobiology of high pathogenicity avian influenza viruses in birds. Avian Dis 51: 242–249.
- 3. Peiris JS, de Jong MD, Guan Y (2007) Avian influenza virus (H5N1): a threat to human health. Clin Microbiol Rev 20: 243–267.
- 4. Garten W, Klenk HD (1999) Understanding influenza virus pathogenicity. Trends Microbiol 7: 99–100.
- 5. Stieneke-Grober A, Vey M, Angliker H, Shaw E, Thomas G, et al. (1992) Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. EMBO J 11: 2407–2414.
- 6. Bosch FX, Orlich M, Klenk HD, Rott R (1979) The structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses. Virology 95: 197–207.
- 7. Horimoto T, Kawaoka Y (1994) Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus. J Virol 68: 3120–3128.
- 8. Senne DA, Panigrahy B, Kawaoka Y, Pearson JE, Suss J, et al. (1996) Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the HA cleavage site as a marker of pathogenicity potential. Avian Dis 40: 425–437.
- 9. Garten W, Klenk H.-D (2008) Cleavage Activation of the Influenza Virus Hemagglutinin and Its Role in Pathogenesis. In: Klenk H-D, Matrosovich , Stech J, editors. Avian Influenza. Basel: Karger. pp. 156–167.
- 10. Kawaoka Y, Webster RG (1985) Evolution of the A/Chicken/Pennsylvania/83 (H5N2) influenza virus. Virology 146: 130–137.
- 11. Horimoto T, Rivera E, Pearson J, Senne D, Krauss S, et al. (1995) Origin and molecular changes associated with emergence of a highly pathogenic H5N2 influenza virus in Mexico. Virology 213: 223–230.
- 12. Rohm C, Horimoto T, Kawaoka Y, Suss J, Webster RG (1995) Do hemagglutinin genes of highly pathogenic avian influenza viruses constitute unique phylogenetic lineages? Virology 209: 664–670.
- 13. Garcia M, Crawford JM, Latimer JW, Rivera-Cruz E, Perdue ML (1996) Heterogeneity in the haemagglutinin gene and emergence of the highly pathogenic phenotype among recent H5N2 avian influenza viruses from Mexico. J Gen Virol 77(Pt 7): 1493–1504.
- 14. Perdue ML, Garcia M, Senne D, Fraire M (1997) Virulence-associated sequence duplication at the hemagglutinin cleavage site of avian influenza viruses. Virus Res 49: 173–186.
- 15. Suarez DL, Senne DA, Banks J, Brown IH, Essen SC, et al. (2004) Recombination resulting in virulence shift in avian influenza outbreak, Chile. Emerg Infect Dis 10: 693–699.
- 16. Pasick J, Handel K, Robinson J, Copps J, Ridd D, et al. (2005) Intersegmental recombination between the haemagglutinin and matrix genes was responsible for the emergence of a highly pathogenic H7N3 avian influenza virus in British Columbia. J Gen Virol 86: 727–731.
- 17. Khatchikian D, Orlich M, Rott R (1989) Increased viral pathogenicity after insertion of a 28S ribosomal RNA sequence into the haemagglutinin gene of an influenza virus. Nature 340: 156–157.
- 18. de Wit E, Munster VJ, van Riel D, Beyer WE, Rimmelzwaan GF, et al. (2009) Molecular determinants of adaptation of HPAI H7N7 viruses to efficient replication in the human host. J Virol.
- 19. Kawaoka Y, Naeve CW, Webster RG (1984) Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? Virology 139: 303–316.
- 20. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, et al. (2004) Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature 431: 703–707.
- 21. Pappas C, Aguilar PV, Basler CF, Solorzano A, Zeng H, et al. (2008) Single gene reassortants identify a critical role for PB1, HA, and NA in the high virulence of the 1918 pandemic influenza virus. Proc Natl Acad Sci U S A 105: 3064–3069.
- 22. Tumpey TM, Maines TR, Van Hoeven N, Glaser L, Solorzano A, et al. (2007) A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission. Science 315: 655–659.
- 23. Matsuoka Y, Swayne DE, Thomas C, Rameix-Welti MA, Naffakh N, et al. (2009) Neuraminidase stalk length and additional glycosylation of the hemagglutinin influence the virulence of influenza H5N1 viruses for mice. J Virol 83: 4704–4708.
- 24. Fernandez-Sesma A, Marukian S, Ebersole BJ, Kaminski D, Park MS, et al. (2006) Influenza virus evades innate and adaptive immunity via the NS1 protein. J Virol 80: 6295–6304.
- 25. Jiao P, Tian G, Li Y, Deng G, Jiang Y, et al. (2008) A single-amino-acid substitution in the NS1 protein changes the pathogenicity of H5N1 avian influenza viruses in mice. J Virol 82: 1146–1154.
- 26. Li Z, Jiang Y, Jiao P, Wang A, Zhao F, et al. (2006) The NS1 gene contributes to the virulence of H5N1 avian influenza viruses. J Virol 80: 11115–11123.
- 27. Lipatov AS, Andreansky S, Webby RJ, Hulse DJ, Rehg JE, et al. (2005) Pathogenesis of Hong Kong H5N1 influenza virus NS gene reassortants in mice: the role of cytokines and B- and T-cell responses. J Gen Virol 86: 1121–1130.
- 28. Seo SH, Hoffmann E, Webster RG (2002) Lethal H5N1 influenza viruses escape host anti-viral cytokine responses. Nat Med 8: 950–954.
- 29. Fouchier RA, Schneeberger PM, Rozendaal FW, Broekman JM, Kemink SA, et al. (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.
- 30. 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.
- 31. de Jong MD, Simmons CP, Thanh TT, Hien VM, Smith GJ, et al. (2006) Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med 12: 1203–1207.
- 32. Gabriel G, Dauber B, Wolff T, Planz O, Klenk HD, et al. (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.
- 33. Salomon R, Franks J, Govorkova EA, Ilyushina NA, Yen HL, et al. (2006) The polymerase complex genes contribute to the high virulence of the human H5N1 influenza virus isolate A/Vietnam/1203/04. J Exp Med 203: 689–697.
- 34. Snyder MH, Buckler-White AJ, London WT, Tierney EL, Murphy BR (1987) The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus polymerase genes each specify attenuation of avian-human influenza A/Pintail/79 reassortant viruses for monkeys. J Virol 61: 2857–2863.
- 35. Baigent SJ, McCauley JW (2001) Glycosylation of haemagglutinin and stalk-length of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture. Virus Res 79: 177–185.
- 36. Banks J, Speidel ES, Moore E, Plowright L, Piccirillo A, et al. (2001) Changes in the haemagglutinin and the neuraminidase genes prior to the emergence of highly pathogenic H7N1 avian influenza viruses in Italy. Arch Virol 146: 963–973.
- 37. Deshpande KL, Naeve CW, Webster RG (1985) The neuraminidases of the virulent and avirulent A/Chicken/Pennsylvania/83 (H5N2) influenza A viruses: sequence and antigenic analyses. Virology 147: 49–60.
- 38. Hoffmann E, Stech J, Leneva I, Krauss S, Scholtissek C, et al. (2000) Characterization of the influenza A virus gene pool in avian species in southern China: was H6N1 a derivative or a precursor of H5N1? J Virol 74: 6309–6315.
- 39. Zhirnov OP, Klenk HD (2009) Alterations in caspase cleavage motifs of NP and M2 proteins attenuate virulence of a highly pathogenic avian influenza virus. Virology 394: 57–63.
- 40. Munier S, Larcher T, Cormier-Aline F, Soubieux D, Su B, et al. (2010) A genetically engineered waterfowl influenza virus with a deletion in the stalk of the neuraminidase has increased virulence for chickens. J Virol 84: 940–952.
- 41. Ma W, Brenner D, Wang Z, Dauber B, Ehrhardt C, et al. (2010) The NS segment of an H5N1 highly pathogenic avian influenza virus (HPAIV) is sufficient to alter replication efficiency, cell tropism, and host range of an H7N1 HPAIV. J Virol 84: 2122–2133.
- 42. Stech O, Veits J, Weber S, Deckers D, Schroer D, et al. (2009) Acquisition of a polybasic hemagglutinin cleavage site by a low-pathogenic avian influenza virus is not sufficient for immediate transformation into a highly pathogenic strain. J Virol 83: 5864–5868.
- 43. Starick E, Beer M, Hoffmann B, Staubach C, Werner O, et al. (2008) Phylogenetic analyses of highly pathogenic avian influenza virus isolates from Germany in 2006 and 2007 suggest at least three separate introductions of H5N1 virus. Vet Microbiol 128: 243–252.
- 44. Weber S, Harder T, Starick E, Beer M, Werner O, et al. (2007) Molecular analysis of highly pathogenic avian influenza virus of subtype H5N1 isolated from wild birds and mammals in northern Germany. J Gen Virol 88: 554–558.
- 45. Stech J, Stech O, Herwig A, Altmeppen H, Hundt J, et al. (2008) Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification. Nucleic Acids Res 36: e139.
- 46. Rott R, Orlich M, Scholtissek C (1976) Attenuation of pathogenicity of fowl plague virus by recombination with other influenza A viruses nonpathogenic for fowl: nonexculsive dependence of pathogenicity on hemagglutinin and neuraminidase of the virus. J Virol 19: 54–60.
- 47. Scholtissek C, Vallbracht A, Flehmig B, Rott R (1979) Correlation of pathogenicity and gene constellation of influenza A viruses. II. Highly neurovirulent recombinants derived from non-neurovirulent or weakly neurovirulent parent virus strains. Virology 95: 492–500.
- 48. Wasilenko JL, Lee CW, Sarmento L, Spackman E, Kapczynski DR, et al. (2008) NP, PB1, and PB2 viral genes contribute to altered replication of H5N1 avian influenza viruses in chickens. J Virol 82: 4544–4553.
- 49. Ito T, Goto H, Yamamoto E, Tanaka H, Takeuchi M, et al. (2001) Generation of a highly pathogenic avian influenza A virus from an avirulent field isolate by passaging in chickens. J Virol 75: 4439–4443.
- 50. Pfeiffer J, Pantin-Jackwood M, To TL, Nguyen T, Suarez DL (2009) Phylogenetic and biological characterization of highly pathogenic H5N1 avian influenza viruses (Vietnam 2005) in chickens and ducks. Virus Res 142: 108–120.
- 51. Kalthoff D, Breithaupt A, Teifke JP, Globig A, Harder T, et al. (2008) Highly pathogenic avian influenza virus (H5N1) in experimentally infected adult mute swans. Emerg Infect Dis 14: 1267–1270.
- 52. Klopfleisch R, Werner O, Mundt E, Harder T, Teifke JP (2006) Neurotropism of highly pathogenic avian influenza virus A/chicken/Indonesia/2003 (H5N1) in experimentally infected pigeons (Columbia livia f. domestica). Vet Pathol 43: 463–470.
- 53. Perkins LE, Swayne DE (2001) Pathobiology of A/chicken/Hong Kong/220/97 (H5N1) avian influenza virus in seven gallinaceous species. Vet Pathol 38: 149–164.
- 54. Stech J, Xiong X, Scholtissek C, Webster RG (1999) Independence of evolutionary and mutational rates after transmission of avian influenza viruses to swine. J Virol 73: 1878–1884.
- 55. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
- 56. Pavlova SP, Veits J, Keil GM, Mettenleiter TC, Fuchs W (2009) Protection of chickens against H5N1 highly pathogenic avian influenza virus infection by live vaccination with infectious laryngotracheitis virus recombinants expressing H5 hemagglutinin and N1 neuraminidase. Vaccine 27: 773–785.
- 57. Alexander DJ (2008) Chapter 2.3.4. Avian influenza. In: Vallat B, editor. Manual of Diagnostic Tests & Vaccines for Terrestrial Animals. 6th ed:. OIE.
- 58. Kalthoff D, Breithaupt A, Helm B, Teifke JP, Beer M (2009) Migratory status is not related to the susceptibility to HPAIV H5N1 in an insectivorous passerine species. PLoS One 4: e6170.