Content area
Highly pathogenic avian influenza (HPAI) H5N1 virus is causing the death of a large number of wild birds and poultry. HPAI H5N1 was reported in the north of Iran in 2011. In this study, two A/Chicken/Iran/271/2011 and A/Duck/Iran/178/2011 viruses were genetically characterized by sequence analysis of Hemagglutinin (HA) and Neuraminidase (NA) genes. Phylogenetic analysis revealed that these viruses were different from previous Iranian isolates (Clade 2.2) and belonged to the subclade 2.3.2.1. The results showed that the detected viruses are almost identical to each other and closely related to HPAI H5N1 strains isolated in Mongolia in 2010. Based on the amino acid sequence analysis, these viruses at their HA cleavage sites contained the multibasic amino acid motif PQRERRRK-R/GLF lacking a lysine residue compared with the previous reports of the same motif. There is also a 20-amino acid deletion (resides 49-69) in the NA stalk similar to other viruses isolated after 2000. It seems that introduction of HPAI H5N1 to Iran might have happened by wild birds from Mongolian origin virus.[PUBLICATION ABSTRACT]
Trop Anim Health Prod (2014) 46:549554 DOI 10.1007/s11250-013-0528-7
REGULAR ARTICLES
Molecular characterization of the surface glycoprotein genes of highly pathogenic H5N1 avian influenza viruses detected in Iran in 2011
Ebrahim Kord & Amir Kaffashi & Hadi Ghadakchi &
Fatemeh Eshratabadi & Zakaria Bameri &
Abdelhamed Shoushtari
Accepted: 18 December 2013 /Published online: 5 January 2014 # Springer Science+Business Media Dordrecht 2014
Abstract Highly pathogenic avian influenza (HPAI) H5N1 virus is causing the death of a large number of wild birds and poultry. HPAI H5N1 was reported in the north of Iran in 2011. In this study, two A/Chicken/Iran/271/2011 and A/Duck/Iran/ 178/2011 viruses were genetically characterized by sequence analysis of Hemagglutinin (HA) and Neuraminidase (NA) genes. Phylogenetic analysis revealed that these viruses were different from previous Iranian isolates (Clade 2.2) and belonged to the subclade 2.3.2.1. The results showed that the detected viruses are almost identical to each other and closely related to HPAI H5N1 strains isolated in Mongolia in 2010. Based on the amino acid sequence analysis, these viruses at their HA cleavage sites contained the multibasic amino acid motif PQRERRRK-R/GLF lacking a lysine residue compared with the previous reports of the same motif. There is also a 20-amino acid deletion (resides 4969) in the NA stalk similar to other viruses isolated after 2000. It seems that introduction of HPAI H5N1 to Iran might have happened by wild birds from Mongolian origin virus.
Keywords Influenza . Avian influenza . H5N1 . Highly Pathogenic Avian Influenza (HPAI)
Introduction
Influenza A virus is a member of Orthomyxoviridae family and is classified into different subtypes on the basis of
Hemagglutinin (HA) and Neuraminidase (NA) glycoproteins (Swayne and Suarez, 2000; Qi et al., 2009). HA binds to host cell receptors and has an important role in determining host tropism, it also contains a cleavage site that must be cleaved by host cell proteases. The sequences of this site affect disease severity. NA is crucial for proper budding and release of progeny virions from the cell surface (Yee et al. 2009; Suguitan et al., 2012).
All subtypes of influenza Aviruses have been isolated from birds. The main natural reservoir for influenza A viruses is believed to be wild aquatic birds (Nguyen et al., 2005; Qi et al., 2009; Jourdain et al. 2010). Depending on their virulence in chicken or turkey, avian influenza viruses are divided into two groups, low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI) A viruses. HPAI viruses have polybasic cleavage site and cause high morbidity and mortality compared with the LPAI viruses (Swayne and Suarez, 2000; Vergara-Alert et al., 2013).
Highly pathogenic avian influenza (HPAI) H5N1 have first been isolated in China in 1996 (Xu et al., 1999). These viruses have spread to other countries and caused numerous deaths in poultry and in wild birds since 2003 (Cattoli et al., 2009; DJ, 2012). HPAI H5N1derived from the goose/Guangdong/1/96 (Gs/GD) lineage has spread to many Asian countries(Peiris et al. 2007; Cattoli et al., 2009). Spread of these viruses has been facilitated by wild bird migration, movement of poultry, and poultry products (Gaidet et al., 2007; Cumming et al. 2011). The spread of HPAI H5N1 into a wide range of avian and some mammalian species facilitates the adaptation of these viruses to human population. The first direct transmission of HPAI H5N1 from poultry to humans occurred in Hong Kong in 1997 (Capua and Alexander, 2002; Peiris et al. 2007). Since then, these viruses have posed a serious threat to
E. Kord : A. Kaffashi : H. Ghadakchi : F. Eshratabadi : Z. Bameri :
A. Shoushtari (*)
Razi Vaccine and Serum Research Institute, Karaj, Iran e-mail: [email protected]
550 Trop Anim Health Prod (2014) 46:549554
humans health. Two outbreaks of HPAI H5N1 have been officially reported in Iran in 2006 and 2008 (Shoushtari et al., 2008; Fereidouni et al., 2010). The re-emergence of these viruses was subsequently reported in 2011. This study investigates molecular properties of two viruses (A/Chicken/ Iran/271/2011 and A/Duck/Iran/178/2011) by nucleotide sequence analysis of the complete HA and NA gene segments.
Materials and methods
Virus detection and RNA extraction Viruses were detected form cloacal swabs obtained from backyard chicken and duck flocks in Mazandaran province in the north part of Iran. These birds were in contact with migratory birds. Viral RNA was extracted using the High Pure Viral RNA kit (Roche, Germany) following the manufacturers instructions. All samples were tested by molecular methods recommended by WHO using RT-PCR targeting the M genes. All samples were tested for H5N1, H9, and H7 influenza viruses.
Primer design Specific primers (Table 1) were designed considering the conserved regions of HA and NA gene segments by Oligo software (National Biosciences, USA).
RT-PCR and DNA sequencing Full-length of HA and NA gene segments as two overlapping fragments was amplified using the One-Step RT-PCR system. Amplifications were performed in 50-l PCR reaction mixtures. Each PCR reaction contained 27.5-l de-ionized water, 10-l PCR buffer, 4-l RNA, 2.5-l DTT, 1-l dNTPs, 1-l High fidelity Expand DNA polymerase, and 2-l of each forward and reverse primers (200 nM/l). Conditions for full HA gene amplification included 45 min at 45 C for RT stage and an initial 3-min denaturation step at 94 C and 35 cycles of denaturation at 94 C for 1 min, annealing at 50 C for 1 min, and extension at 68 C for 5 min. An additional extension step at 68 C for 10 min was done to complete the amplification. The amplification of NA differed only in
extension time, which was 4 min. Amplified RT-PCR products were electrophoresed on the 1.5 % low melting point (LMP) agarose gel. The distinct bands were purified from gel using a High Pure PCR Product Purification kit (Roche, Germany). The purified DNA fragments were sequenced from both directions using cycle sequencing dye terminator chemistry (Applied Biosystems).
Phylogenetic analysis BioEdit software was used for the assembly and translation of nucleotide sequences into protein sequences. Multiple nucleotide and amino acid sequence alignments were performed using Megalign MEGA5 package (MEGA5 1993-2011). MEGA5 was also used for the construction of phylogenetic trees using the distance-based Neighbor-Joining method with 1000 bootstrap replicates. Phylogenetic trees generated for this study were based on full or nearly full length HA and NA gene open reading frames (ORF). Clades were classified according to the WHO nomenclature( WHO/OIE/FAO H5N1 Evolution Working Group, 2012).
Results
Field observations The dead birds had facial edema, swollen cyanotic comb and wattles, and diffused hemorrhage in many organs such as cerebellum, heart, lung, rectum, and liver. There were also variable lesions like edema and congestion of lung, focal necrosis within the parenchyma of pancreas, and enlargement of spleen and liver. A few birds showed exudates around their nostril.
Phylogenetic analysis The phylogenetic analysis of HA and NA genes showed that the gene segments of our study were of avian origin. The results revealed that similar genes in chickens and ducks were almost identical to each other (>99 %) and were closely related to A/whooper swan/Mongolia/1/2010 Mongolian isolate in Clade 2.3.2.1. Phylogenetic analysis of the NA showed a similar relationship to that observed for the HA (Figs. 1 and 2).
Molecular characterization Based on the amino acid sequence analysis, the detected viruses had a multibasic amino acid motif PQRERRRK-R/GLF at their HA cleavage site. The HA gene obtained from both chicken and duck samples had a D94N (Asp94Asn (mutation (H5 numbering). Potential glycosylation sites for HA and NA with the N-X-T/S motif were identified. Six potential N-glycosylation sites were found in the HA protein sequences that all were same to the reference virus and the previous report with exception of NSS motif in the position 140142. For NA, the viruses have the same N-glycosylation sites to the reference virus with the exception of those sites located in deleted motif in the stalk.
Table 1 Specific primer for HA and NA segment genes used for avian influenza viruses RT-PCR
Primers Sequences
H5HA-F1 5-CAA AAT GGA GAA AAT AGT GC-3 H5HA-R1 5-AGA GGG TGT ATG TTG TGG-3 H5HA-F2 5-AGT GGA ATA TGG TAA CTG C-3 H5HA-R1 5-AGA GGG TGT ATG TTG TGG-3 H5NA-F1 5-CAG AAG ATA ATA ACC ATT GG-3 H5NA-R1 5-TCT TAT ATG ATG CCT GTC C-3 H5NA-F2 5-AGA CAC TAT CAA GAG TTG G-3 H5NA-R2 5-ACT TGT CAA TGG TGA ATG G-3
Trop Anim Health Prod (2014) 46:549554 551
72
Fig. 1 Phylogenetic treeof HA of the H5N1 influenzaA viruses isolated in Iran in 2011. The tree was generated by the distance-based Neighbor-Joining method in software MEGA 5. The reliability of the tree was assessed by bootstrap analysis with 1,000 replicates
A/chicken/Brasov/RO-AI-350/2006
A/chicken/Nigeria/08RS848-45/2006
A/chicken/Romania/3293/05
A/Bar-headed Goose/Qinghai/65/05
A/Bar-headed Goose/Qinghai/68/05
A/swan/England/26-70/2008
A/chicken/India/NIV33487/06
A/Cygnus cygnus/Iran/754/2006
A/falcon/Saudi Arabia/D1795/2005
A/chicken/Nigeria/228-5/2006
A/Iraq/754/2006
A/Turkey/12/2006
56
21
24
95
2.2
100
79
93
93
60
60
97
2.4
A/chicken/Yunnan/207/2004
A/chicken/Bangli Bali/BPPV6-2/20042.1.3
A/Indonesia/CDC1032/2007
A/chicken/Indonesia/CDC24/2005
A/Chicken/Sembawa/BPPV-III/2005
84 60
100
100
78
2.1
2.1
100
A/chicken/Hong Kong/3123/1/2002
1
A/Hong Kong/213-DkPass/2003
A/chicken/Guangxi/1951/2006
2.3.4
100
94
A/chicken/Vietnam/NCVD-40/2007
A/Muscovy duck/Vietnam/56/2007
A/duck/Thai Ninh/07-86/2007
A/chicken/Bac Giang/07-74/2007
A/Muscovy duck/Vietnam/48/2007
A/duck/Ha Tinh/07-53/2007
45
100
2.3
100
48
2.3
A/goose/Yunnan/3720/2005
A/large billed crow/Hong Kong/885/2009
A/Duck/Iran/178/2011
A/Chicken/Iran/271/2011
A/whooper swan/Mongolia.8/2009
A/whooper swan/Mongolia/11/2010
A/whooper swan/Mongolia/1/2010
A/bar-headed goose/Mongolia/X53/2009
A/common goldeneye/Mongolia/X60/2009
2.3.2.1
98
100
100
56
91
100
64
58
NA amino acid sequences analysis revealed that detected viruses contained a 20-amino acid deletion (resides 4969) in the stalk of NA glycoprotein. From those amino acid mutations with potential to confer viral resistance to NA inhibitors, only H274Y mutation was observed.
Discussion
The highly pathogenic avian influenza (H5N1) was first identified in China in 1996 (Xu et al., 1999). Since its re-emergence in 2003, HPAI H5N1 has been reported from many countries, causing outbreaks in poultry and wild birds (Cattoli et al., 2009; Brown, 2010). These viruses were first isolated from dead wild birds in the north of Iran in 2006 (Shoushtari et al., 2008). The north of Iran in the southern part of Caspian Sea consists of 700 km of sandy shoreline, freshwater lakes,
marshes, and brackish lagoons in the central Gilan province, Gorgan Bay, and Turkman steppes, which provide a favorable environment for breeding and wintering of waterfowls. During the spring and autumn migration seasons, a large number of shorebirds pass through the south Caspian region on their way between breeding grounds in the Arctic and wintering grounds in the Persian Gulf and eastern and southern Africa. It is generally accepted that the migratory birds are the major spreading agent of avian influenza (Hagemeijer, 2006). Subsequently, two outbreaks of HPAI H5N1 were officially reported from the north of Iran in 2008 (Fereidouni et al., 2010) and 2011(Dr Seyed Mohsen Dastoor, 2011), showing this region as the main entrance for these viruses to the country.
Influenza A viruses have a capacity of mutating and evolving over time, changing their genetic and antigenic characteristics (Lauring and Andino, 2010). The continuing circulation
552 Trop Anim Health Prod (2014) 46:549554
A/duck/Phu Tho/07-48/2007
2.3.2
2.3.4.3
A/duck/Lang Son201/2005
2.2.1
A/Cygnus olor/Czech Republic/5170/20065
A/goose/Guangxi/2112/2004
A/chicken/Hong Kong/947/2006
A/human/China/GD02/2006
2.3.4
A/chicken/Bali/UT2091/2005
A/chicken/Guiyang/2147/2005
A/goose/Yunnan/4129/2005
A/goose/Yunnan/3720/2005
A/duck/Guangxi/89/2006
A/Muscovy duck/Vietnam/56/2007
A/goose/Guangxi/1458/2006
A/chicken/Guangxi/1951/2006
2.1
2.3
A/falcon/Saudi Arabia/D1795/2005
A/chicken/Nigeria/228-5/2006
A/Iraq/755/2006
2.2
A/chicken/Korea/es/2003
9
2.5
A/migratory duck/Jiangxi/1653/2005
A/chicken/Vietnam/24/2004
A/chicken/Thailand/PC-170/2006
1
A/chicken/Guiyang/441/2006
7
4
A/chicken/Xinjiang/68/2005
A/large billed crow/Hong Kong/885/2009
A/chicken/Iran/271/2011
A/duck/Iran/178/2011
A/whooper swan/Mongolia/21/2010
A/bar-headed goose/Mongolia/X54/2009
2.3.2.1
Fig. 2 Phylogenetic tree of NA of the H5N1 influenza A viruses isolated in Iran in 2011. The tree was generated by the distance-based Neighbor-Joining method in software MEGA 5. The reliability of the tree was assessed by bootstrap analysis with 1,000 replicates
of HPAI H5N1 in different geographical regions and hosts has led to an increase in the number of isolates that have been genetically sequenced. As a consequence, several distinct genetic groups or clades have been identified. During 2005 2008 period, clade 2.2 was dominant in Asia (Cattoli et al., 2009). But clade 2.3.2.1 has been found increasingly in poultry and wild birds in several Asian countries since 2007(Peiris, De Jong, and Guan, 2007; DJ, 2012). The results of this study showed that our detected viruses genetically belong to clade2.3.2.1, and the previously dominant clade 2.2 (that isolated in 2006) (Shoushtari et al., 2008; Fereidouni et al., 2010) has been replaced by this clade. Phylogenetic analysis showed the major homology between Iranian recent isolates and Mongolian ones, suggesting that HPAI viruses have been introduced to Iran by wild migratory birds, perhaps from Mongolian virus origin.
HA mediates virus binding to cell receptors and facilitates membrane fusion between viral envelope and cell endosomal membrane. HA plays an important role in determining the systemic spread and pathogenicity of avian influenza viruses (Ito et al., 1998; Schrauwen et al., 2012). LPAI viruses have a single basic amino acid in their HA cleavage site that cleaved only by Tripsin-like proteases. Therefore, these viruses are usually limited to respiratory or intestinal tract. In contrast, HPAI viruses have polybasic amino acid motif in their HA cleavage site that cleaved by ubiquitous proteases and can cause systemic disease (Swayne and Suarez, 2000; Bogs et al., 2010). The multiple basic amino acid motif at the HA cleavage site is essential for lethal infection in chicken. The HA cleavage site of our detected viruses had such motif
(PQRERRR-KR/GLF), characteristic of highly pathogenic avian influenza strains, but it was different from that of the previous Iranian isolates, as a residue (lysine) was lacking (Shoushtari et al., 2008). These results were consistent with histopathological findings and the observation of extensive gross lesions especially in vital organs such as brain and heart of dead chickens in which the viruses were detected from. It has been previously shown that the poultry including ducks, chickens, pheasants, herons, geese, and quails are the host of the viruses containing the RERRR-KR motif, but the effect of the deletion of one residue in the multiple basic motif on the host range is not known (Matrosovich et al., 1999; Li et al., 2004).
Receptor binding is a major factor for determining host species tropism. Avian influenza viruses bind to sialic acid (SA)a-2, 3-Gal receptors, whereas human influenza viruses prefer SA-a-2, 6-Gal receptors (Lin and Cannon, 2002; Stevens et al., 2006). The HA molecules in our study share the same amino acids Q222 (position 226 in H3 numbering) and G224 (position 228 in H3 numbering) as avian viruses are related to receptor binding. Other relavant amino acid residues were identical to those of A/Hong Kong/156/97and A/Goose/Guangdong/1/96 viruses with the exception of S129L and S133A substitutions.
There are some mutations such as Q222L, G224S, N182 K Q192R D94N that enhance the binding of H5N1 viruses to the sialic acid (SA) a2, 6-Gal receptors (Su et al., 2008). Our detected viruses had only D94N mutation, which contributes to the structural changes and indirectly influences the HA receptor interaction considering that residue 94 is located close to the 220 loop of the receptor binding domain (Lin and Cannon, 2002; Stevens et al., 2006).
N-Glycosylation sites of HA and NA have been found to play a vital role in receptor binding, infectivity, host immune responses, virus release, and neurovirulence. During viral evolution, N-Glycosylation sites are easily changed and may be deleted or added. So these potential modifications can alter the complexity of viral glycoproteins and increase or decrease virus transmissibility, replication, and infectivity (Vigerust and Shepherd, 2007; Das et al., 2011). For HA protein sequences, detected viruses same as Mongolian isolate had six potential N-glycosylation sites that are more than those of A/Goose/Guangdong/1/96. It is known that carbohydrate addition can have both positive and negative effects on the virus (Vigerust and Shepherd, 2007). The study of pathogencity of these viruses that has several carbohydrate additions may be beneficial.
NA is one of the two major glycoproteins on the virus surface. The NA plays a central role in the release of the virus from infected cells by removing terminal sialic acids from oligosaccharide side chains to which the viral HA binds (Yee et al. 2009; Wu et al., 2010). The functional balance between HA receptor affinity for virus attachment and NA sialidase
Trop Anim Health Prod (2014) 46:549554 553
activity for release is necessary for efficient influenza virus replication. The NA stalk-motif played a critical role in virulence and pathogenesis of H5N1 avian influenza virus (Yee et al. 2009). The NA stalk region varies considerably among different viruses. Since 2000, a new special NA stalk-motif had been observed in H5N1 influenza viruses, with a 20-amino acid deletion (positions 4969) in the stalk region (Baigent and McCauley, 2001; Zhou et al., 2009). There is interdependence between HA glycosylation and NA stalk length (Wagner et al., 2000; Lu et al., 2005). A virus containing HAwith little carbohydrate can tightly bind to the receptor requiring greater NA activity for particle release. Conversely, an HA with more extensive glycosilation interacts weakly with receptors and requires a less NA activity. Previous study showed that H5N1 influenza viruses with a 20-amino acid deletion in the stalk region have glycosylation at aa170 or aa169 that decreases HA binding affinity for sialic acids (Zhou et al., 2009). So, the deficiency in NA activity conferred by the shortened protein stalk is compensated by the decrease of HA receptor-binding affinity. The viruses in our study have a potential site for glycosilation at position 165167 (aa166). Considering that there is a 20-amino acid deletion in NA stalk, whether this site is acting as aa170 should be investigated. Some studies have suggested that the deletion in the NA stalk may be associated with adaptation of influenza viruses to land-based poultry and increased virulence and pathogenesis in poultry and mammalian (Matrosovich et al., 1999; Li et al., 2004) This study revealed that the detected viruses had this special deletion, which is likely an important factor for emergence of H5N1 isolates with increased virulence since 2000.
Antiviral compounds are needed for influenza treatment during a severe outbreak. NA is an important target for some drugs such as Oseltamivir and Zanamivir. A great concern about available antivirals is the risk of development of drug resistance. The most important mutations regarding drug resistance include E119V, R293K, H274Y, and N294S (De Jong et al., 2005; Le et al., 2005; Qi et al., 2009; Tisoncik-Go, Cordero, and Rong, 2013). One of the changes that was observed in Oseltamivir-treated patients infected with the H5N1 HPAI strains is H274Y mutation (Ho et al., 2007; Ferraris and Lina, 2008). Our detected viruses have also had the mutation H274Y indicating that they possess some degree of resistance to Oseltamivir. This is an unfavorable observation given the pandemic potential and extremely virulent nature of these viruses.
In conclusion, the Iranian detected HPAI H5N1 viruses in 2011 that belong to clade 2.3.2.1 and were different from previous reports. Our findings suggest these viruses were introduced into the country by wild birds likely from Mongolia. Therefore, the continuous monitoring both in poultry and in wild birds especially in the north part of Iran is necessary to prevent more outbreaks.
Acknowledgments This study was supported by Razi Vaccine and Serum Research Institute. We would like to thank M. Mahmoodzadeh and the other staff of the Department of Avian Diseases in the institute.
References
Baigent, S. J., and McCauley, J. W. (2001). Glycosylation of haemagglutinin and stalk-length of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture. Virus research 79(1), 177-185.
Bogs, J., Veits, J., Gohrbandt, S., Hundt, J., Stech, O., Breithaupt, A.,
Teifke, J. P., Mettenleiter, T. C., and Stech, J. (2010). Highly pathogenic H5N1 influenza viruses carry virulence determinants beyond the polybasic hemagglutinin cleavage site. PLoS One 5(7), e11826. Brown, I. H. (2010). Summary of avian influenza activity in Europe,
Asia, and Africa, 2006-2009. Avian diseases 54(s1), 187-193. Capua, I., and Alexander, D. J. (2002). Avian influenza and human health.
Acta tropica 83(1), 1-6.
Cattoli, G., Fusaro, A., Monne, I., and Capua, I. (2009). H5N1 Virus
Evolution in EuropeAn Updated Overview. Viruses 1(3), 1351-1363.
Cumming, G. S., Caron, A., Abolnik, C., Cattoli, G., Bruinzeel, L. W.,
Burger, C. E., Cecchettin, K., Chiweshe, N., Mochotlhoane, B., and Mutumi, G. L. (2011). The ecology of influenza A viruses in wild birds in southern Africa. EcoHealth 8(1), 4-13.
Das, S. R., Hensley, S. E., David, A., Schmidt, L., Gibbs, J. S., Puigb, P.,
Ince, W. L., Bennink, J. R., and Yewdell, J. W. (2011). Fitness costs limit influenza A virus hemagglutinin glycosylation as an immune evasion strategy. Proceedings of the National Academy of Sciences 108(51), E1417-E1422.
De Jong, M. D., Thanh, T. T., Khanh, T. H., Hien, V. M., Smith, G. J. D.,
Chau, N. V., Cam, B. V., Qui, P. T., Ha, D. Q., and Guan, Y. (2005). Oseltamivir resistance during treatment of influenza A (H5N1) infection. New England Journal of Medicine 353(25), 2667-2672. DJ, S. (2012). Avian influenza and the implication for human infection.
Microbiology 33, 172-173.
Dr Seyed Mohsen Dastoor, H. o. I. V. O., Iran Veterinary Organization,
Ministry of Jihad-e-Agriculture, Tehran, Iran (15-OCT-2011). Avian influenza (62): Iran (Mazandaran), H5N1 - OIE. In A ProMED-mail post -OIE, Vol. 62. http://thisbluemarble.com/showthread.php?p=317632
Web End =http://thisbluemarble.com/showthread. http://thisbluemarble.com/showthread.php?p=317632
Web End =php?p=317632 .
Fereidouni, S. R., Werner, O., Starick, E., Beer, M., Harder, T. C.,
Aghakhan, M., Modirrousta, H., Amini, H., Moghaddam, M. K., and Bozorghmehrifard, M. H. (2010). Avian influenza virus monitoring in wintering waterbirds in Iran, 2003-2007. Virol J 7(43), 1754-1757.
Ferraris, O., and Lina, B. (2008). Mutations of neuraminidase implicated in neuraminidase inhibitors resistance. Journal of Clinical Virology 41(1), 13-19.
Gaidet, N., Dodman, T., Caron, A., Balana, G., Desvaux, S., Goutard, F.,
Cattoli, G., Martin, V., Tripodi, A., and Lamarque, F. (2007). Influenza surveillance in wild birds in Eastern Europe, the Middle East, and Africa: Preliminary results from an ongoing FAO-led survey. Journal of Wildlife Diseases 43(3 Supplement), S22-S28. Hagemeijer, W. a. T. M. (2006). Migratory flyways in Europe, Africa and
Asia and the spread of HPAI H5N1. in International Scientific Conference On Avian Influenza and Wild Birds,FAO and OIE, Rome, Italy. http://www.fao.org/ag/againfo/subjects/en/health/diseases-cards/conference/documents/hagemeijer-mundkur.pdf
Web End =http://www.fao.org/ag/againfo/subjects/en/health/ http://www.fao.org/ag/againfo/subjects/en/health/diseases-cards/conference/documents/hagemeijer-mundkur.pdf
Web End =diseases-cards/conference/documents/hagemeijer-mundkur.pdf Ho, H. T., Hurt, A. C., Mosse, J., and Barr, I. (2007). Neuraminidase inhibitor drug susceptibility differs between influenza N1 and N2 neuraminidase following mutagenesis of two conserved residues. Antiviral research 76(3), 263-266.
554 Trop Anim Health Prod (2014) 46:549554
Ito, D., Imai, Y., Ohsawa, K., Nakajima, K., Fukuuchi, Y., and Kohsaka,S. (1998). Microglia-specific localisation of a novel calcium binding protein, Iba1. Molecular brain research 57(1), 1-9.
Jourdain, E., Gunnarsson, G., Wahlgren, J., Latorre-Margalef, N., Brjer,C., Sahlin, S., Svensson, L., Waldenstrm, J., Lundkvist, ., and Olsen, B. (2010). Influenza virus in a natural host, the mallard: experimental infection data. PLoS One 5(1), e8935.
Lauring, A. S., and Andino, R. (2010). Quasispecies theory and the behavior of RNA viruses. PLoS pathogens 6(7), e1001005.
Le, Q. M., Kiso, M., Someya, K., Sakai, Y. T., Nguyen, T. H., Nguyen, K.H. L., Pham, N. D., Ngyen, H. H., Yamada, S., and Muramoto, Y. (2005). Avian flu: isolation of drug-resistant H5N1 virus. Nature 437(7062), 1108-1108.
Li, K., Guan, Y., Wang, J., Smith, G., Xu, K., Duan, L., Rahardjo, A., Puthavathana, P., Buranathai, C., and Nguyen, T. (2004). Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 430(6996), 209-213.
Lin, A. H., and Cannon, P. M. (2002). Use of pseudotyped retroviral vectors to analyze the receptor-binding pocket of hemagglutinin from a pathogenic avian influenza A virus (H7 subtype). Virus research 83(1), p. 43-56.
Lu, B., Zhou, H., Ye, D., Kemble, G., and Jin, H. (2005). Improvement of influenza A/Fujian/411/02 (H3N2) virus growth in embryonated chicken eggs by balancing the hemagglutinin and neuraminidase activities, using reverse genetics. Journal of virology 79(11), 6763-6771.
Matrosovich, M., Zhou, N., Kawaoka, Y., and Webster, R. (1999). The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. Journal of virology 73(2), 1146-1155.
Nguyen, D. C., Uyeki, T. M., Jadhao, S., Maines, T., Shaw, M., Matsuoka, Y., Smith, C., Rowe, T., Lu, X., and Hall, H. (2005). Isolation and characterization of avian influenza viruses, including highly pathogenic H5N1, from poultry in live bird markets in Hanoi, Vietnam, in 2001. Journal of virology 79(7), 4201-4212.
Peiris, J. S. M., De Jong, M. D., and Guan, Y. (2007). Avian influenza virus (H5N1): a threat to human health. Clinical Microbiology Reviews 20(2), 243-267.
Qi, X., Li, X., Rider, P., Fan, W., Gu, H., Xu, L., Yang, Y., Lu, S., Wang,H., and Liu, F. (2009). Molecular characterization of highly pathogenic H5N1 avian influenza Aviruses isolated from raccoon dogs in China. PLoS One 4(3), e4682.
Schrauwen, E. J., Herfst, S., Leijten, L. M., van Run, P., Bestebroer, T.M., Linster, M., Bodewes, R., Kreijtz, J. H., Rimmelzwaan, G. F., and Osterhaus, A. D. (2012). The multibasic cleavage site in H5N1 virus is critical for systemic spread along the olfactory and hematogenous routes in ferrets. Journal of virology 86(7), 3975-3984. Shoushtari, A., Hablolvarid, M., Vascellari, M., and Hedayati, A. (2008).
Mortality of wild swans associated with naturally infection with highly pathogenic H5N1 avian influenza virus in Iran. Archives of Razi 62(4), 207-213.
Stevens, J., Blixt, O., Tumpey, T. M., Taubenberger, J. K., Paulson, J. C., and Wilson, I. A. (2006). Structure and receptor specificity of the
hemagglutinin from an H5N1 influenza virus. science 312(5772), 404-410.
Su, Y., Yang, H. Y., Zhang, B. J., Jia, H. L., and Tien, P. (2008). Analysis of a point mutation in H5N1 avian influenza virus hemagglutinin in relation to virus entry into live mammalian cells. Archives of virology 153(12), 2253-2261.
Suguitan, A. L., Matsuoka, Y., Lau, Y.-F., Santos, C. P., Vogel, L., Cheng, L. I., Orandle, M., and Subbarao, K. (2012). The multibasic cleavage site of the hemagglutinin of highly pathogenic A/Vietnam/1203/2004 (H5N1) avian influenza virus acts as a virulence factor in a host-specific manner in mammals. Journal of virology 86(5), 2706-2714.
Swayne, D., and Suarez, D. (2000). Highly pathogenic avian influenza. Revue scientifique et technique (International Office of Epizootics) 19(2), 463.
Tisoncik-Go, J., Cordero, K. S., and Rong, L. (2013). Analysis of oseltamivir resistance substitutions in influenza virus glycoprotein neuraminidase using a lentivirus-based surrogate assay system. Virologica Sinica, 1-11.
Vergara-Alert, J., Moreno, A., Zabala, J. G., Bertran, K., Costa, T. P.,
Cordn, I., Rivas, R., Maj, N., Busquets, N., and Cordioli, P. (2013). Exposure to a Low Pathogenic A/H7N2 Virus in Chickens Protects against Highly Pathogenic A/H7N1 Virus but Not against Subsequent Infection with A/H5N1. PLoS One 8(3), e58692. Vigerust, D. J., and Shepherd, V. L. (2007). Virus glycosylation: role in virulence and immune interactions. Trends in microbiology 15(5), 211-218.
WHO/OIE/FAO H5N1 Evolution Working Group (2012). Continued evolution of highly pathogenic avian influenza A (H5N1): Updated nomenclature. Influenza and Other Respiratory Viruses 6, 15. doi:http://dx.doi.org/10.1111/j.1750-2659.2011.00298.x
Web End =10.1111/j.1750-2659.2011.00298.x Available from http://onlinelibrary.wiley.com/doi/10.1111/j.1750-2659.2011.00298.x/abstract
Web End =http:// http://onlinelibrary.wiley.com/doi/10.1111/j.1750-2659.2011.00298.x/abstract
Web End =onlinelibrary.wiley.com/doi/10.1111/j.1750-2659.2011.00298.x/ http://onlinelibrary.wiley.com/doi/10.1111/j.1750-2659.2011.00298.x/abstract
Web End =abstract .
Wagner, R., Wolff, T., Herwig, A., Pleschka, S., and Klenk, H. D. (2000). Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics. Journal of virology 74(14), 6316-6323.
Wu, J., Zhang, F., Wang, M., Xu, C., Song, J., Zhou, J., Lin, X., Zhang,Y., Wu, X., and Tan, W. (2010). Characterization of neuraminidases from the highly pathogenic avian H5N1 and 2009 pandemic H1N1 influenza A viruses. PLoS One 5(12), e15825.
Xu, X., Subbarao, K., Cox, N. J., and Guo, Y. (1999). Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology 261(1), 15-19.
Yee, K. S., Carpenter, T. E., and Cardona, C. J. (2009). Epidemiology of H5N1 avian influenza. Comparative immunology, microbiology and infectious diseases 32(4), 325-340.
Zhou, H., Yu, Z., Hu, Y., Tu, J., Zou, W., Peng, Y., Zhu, J., Li, Y., and Zhang, A. (2009). The special neuraminidase stalk-motif responsible for increased virulence and pathogenesis of H5N1 influenza A virus. PLoS One 4(7), e6277.
Springer Science+Business Media Dordrecht 2014