Abstract
In the US, the isolation of H5 subtype avian influenza (AI) viruses has been uncommon in commercial chickens and turkeys, although sporadic isolations have been made from the live bird markets or its supply chain since 1986. In 2002, two different outbreaks of H5 AI occurred in commercial chicken or turkey operations. The first occurred in Texas and was identified as a H5N3 subtype AI virus. The second outbreak was caused by a H5N2 virus isolated from a turkey farm in California. In this study we analyzed recent H5 subtype AI viruses from different avian species and different sources in the US. Most recent H5 subtype isolates shared a high sequence identity and phylogenetically assorted into a separate clade from the Pennsylvania/83 lineage isolates. However, no established lineage was found within this clade and the recent H5 subtype isolates seemed to be the result of separate introductions from the wild bird reservoir. The Texas H5N3 isolate shared the lowest homology with the other recent isolates in the haemagglutinin gene and had a unique haemagglutinin cleavage site sequence of REKR/G (other recent isolates have the typical avirulent motif, RETR/G). Furthermore, this isolate had a 28 amino acid deletion in the stalk region of the neuraminidase protein, a common characteristic of chicken adapted influenza viruses, and may indicate that this virus had actually been circulating in poultry for an extended period of time before it was isolated. In agreement with genetic evidence, the Texas H5N3 isolate replicated better than other H5 isolates in experimentally infected chickens. The outbreak in Texas with a more chicken-adapted H5N3 virus underscores the importance of ongoing surveillance and control efforts regarding the H5 subtype AI virus in the US.
Introduction
Type A influenza viruses infect a variety of avian and mammalian hosts and can cause severe disease in many species. The virus has eight different segments of single-stranded, negative-sense RNA that encode a minimum of 10 proteins. The haemagglutinin (HA) and neuraminidase (NA) surface glycoproteins are divided into 15 and nine different subtypes, respectively. HA functions as the receptor-binding and membrane fusion glycoprotein in cell entry, and NA functions as the receptor-destroying enzyme in virus release. The importance of balanced HA and NA activities for efficient replication of influenza virus have been described (Mitnaul et al., Citation2000; Wagner et al., Citation2000). All 15 HA and nine NA subtypes have been isolated from aquatic birds that are believed to be a natural host and reservoir for influenza viruses. Poultry, specifically chickens and turkeys, are not considered to be a normal host for the virus, although transmission from wild birds to poultry occurs routinely. Although most HA subtypes have been found in poultry, particular emphasis is placed on the H5 or H7 HA subtypes of avian influenza (AI) virus because only these subtypes are known to cause highly pathogenic AI (HPAI) in poultry.
Several outbreaks of HPAI have occurred in North America involving the H5 subtype virus. In 1966, the H5N9 subtype virus, A/Turkey/Ontario/7732/66, caused a limited outbreak (Lang et al., Citation1968). In 1983, HPAI occurred in commercial poultry in Pennsylvania with devastating effects on the poultry industry (Eckroade & Bachin, Citation1987). More recently, HPAI occurred in central Mexico during 1994 to 1995 (Senne et al., Citation1996a). The Pennsylvania 83 and Mexican 94/95 outbreaks clearly demonstrate the emergence of HP H5 AI virus from low pathogenic (LP) precursor H5 AI viruses (Kawaoka et al., Citation1984; Garcia et al., Citation1996), and several different mechanisms of mutation regarding the emergence of HPAI viruses have been documented (Perdue et al., Citation1996; Swayne et al., Citation1998; Suarez et al., Citation2004).
Live bird markets (LBM) provide a favorable environment for mutation of AI virus due to the dynamics of these markets, and progressive genetic drift has been demonstrated in the HA gene of H5 and H7 viruses from LBMs (Suarez et al., Citation1999; Suarez & Senne, Citation2000; Spackman et al., Citation2003). In the LBMs located in the northeastern (NE) US, the best studied LBM system, a variety of avian species including chickens, turkeys, guinea fowl, quail, ducks, and so on, are sold in relatively high numbers with new birds being introduced daily. Retrospective genetic analysis of H7N2 and H5N2 isolates from LBMs provided evidence that the LBMs were the probable source of infection for influenza outbreaks in commercial poultry operations (Suarez et al., Citation1999; Suarez & Senne, Citation2000). Since the LBMs were first identified as a niche for influenza, increased surveillance has demonstrated the sporadic presence of H5N2 influenza viruses in the NE LBMs since 1986 (Senne et al., Citation1992, Citation1996b; Panigrahy et al., Citation2002). The H5N2 viruses, first identified in the LBMs in 1986, were shown to be related to the Pennsylvania 83 HPAI viruses and were endemic in the LBM system until they were finally eradicated in 1989. A separate H5N2 lineage virus was introduced into the NE LBM system in 1993, but it was also eradicated (Horimoto & Kawaoka, Citation1995). After 1993, the number of H5 isolations decreased compared with the previous 8 years. This coincided with the increased rate of isolation of the H7 subtype AI virus. Most H5 isolates after 1993 had characteristics analogous to those of avirulent wild bird H5 viruses. Isolation of H5 subtype AI virus in chickens and turkeys has been rare since 1993.
In 2002, two different outbreaks of H5 occurred in chickens and turkeys. The first outbreak was in Texas and the causative virus was identified as a H5N3 subtype AI virus. The second outbreak was with a H5N2 virus isolated from a turkey farm in California. In the present paper, we characterized these isolates genetically and compared them with other recent non-chicken and non-turkey origin H5 subtype viruses from North America. Furthermore, the replication competency and potential pathogenicity of recent H5 subtype viruses in chickens were assessed.
Materials and Methods
Viruses
Virus isolates for this study were obtained from the National Veterinary Services Laboratories in Ames, Iowa, the Texas Veterinary Medical Diagnostic Laboratory in Gonzales, Texas, the California Animal Health and Food Safety Laboratory System in Fresno, California, and the Department of Medical Microbiology, University of Georgia. All H5 viruses were isolated from domesticated birds except one isolate, A/Ruddy Turnstone/NJ/2242/00. Viruses were received in allantoic fluid after passage in embryonating chicken eggs (ECE). Isolates were passaged one or two additional times at the Southeast Poultry Research Laboratory to make working stocks of the virus. The isolates used in this study are presented in .
Table 1. Avian influenza virus isolates examined in this study
Pathogenicity in chickens
Eight 6-week-old to 8-week-old chickens derived from a specific pathogen free (SPF) flock were inoculated intravenously with 0.2 ml of a 1:10 dilution of bacteria-free allantoic fluid containing AI virus. The chickens were observed daily for illness or death for 10 days post-inoculation (d.p.i.).
Fourteen-day-old ECE passage system
Since all the recent isolates had the same HA cleavage site sequence motif except Texas H5N3 isolates (A/CK/TX/167280-4/02), two H5N2 (A/Pheasant/NJ/1355/98 and A/Avian/NY/31588-3/00) isolates with RETR/G motif and a Texas H5N3 isolate with REKR motif of first passage in 10-day ECE were used as the parent viruses. Viruses were passaged through a 14-day chicken embryo laboratory system that favors the emergence of HP derivatives (Brugh & Beck, Citation1992). Briefly, these AI viruses were inoculated into the allantoic sac of 14-day ECE. Allantoic fluid was collected from all embryos that died in the first 3 days and was examined in a preliminary screening test to identify AI virus derivatives with increased cytopathic effect in trypsin-free chicken embryo fibroblast cultures as compared with the parent AI viruses. Those with increased cytopathic effect on the preliminary screening tests were further examined for high plaquing efficiency in chicken embryo fibroblast cultures in the presence or the absence of trypsin. AI viruses with the ability to produce a cytopathic effect in chicken embryo fibroblast in the absence of trypsin are known to be associated with HP H5 and H7 subtype viruses (Bosch et al., Citation1979).
RNA extraction and sequencing of influenza virus genes
RNA from the isolates used in this study was extracted with the RNeasy mini kit (Qiagen, Valencia, CA, USA) from infected allantoic fluid from ECE. A modified protocol for fluid samples was previously described (Spackman et al., Citation2002). The entire coding region of HA, NA, matrix (MA) , and non-structural (NS) genes were amplified by standard reverse transcription-polymerase chain reaction (RT-PCR) with the Qiagen one-step RT-PCR kit and primers directed to the 12 or 13 conserved bases at the ends of each influenza viral RNA segment. The RT-PCR conditions and primer sequences have been previously described (Suarez et al., Citation1998). The PCR product was separated on an agarose gel by electrophoresis, and amplicons of the appropriate size were subsequently excised from the gel and extracted with the Qiagen gel extraction kit. Sequencing was performed with the PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Perkin-Elmer, Foster City, CA, USA) run on a 3700 automated sequencer (Perkin-Elmer).
Nucleotide and amino acid sequence phylogenetic analysis
The nucleotide sequences were compared initially with the Megalign program (DNASTAR, Madison, WI, USA) using the Clustal V alignment algorithm. Pairwise sequence alignments were also performed in the Megalign program to determine nucleotide and amino acid sequence similarity. Phylogenetic comparisons of the aligned sequence for each gene segment were generated using the maximum parsimony method with 100 bootstrap replicates in a heuristic search using the PAUP 4.0b10 software (Sinauer Associates, Inc, Sunderland, MA, USA) (Swofford, Citation1998).
Experimental chicken infection
Three-week-old SPF White Rock chickens in groups of 10, were inoculated intranasally with one of five AI viruses (). Tracheal and cloacal swabs were collected at 3 and 7 d.p.i. using sterile cotton-tipped applicators (REF 25-806; Hardwood Products Company, Guilford, Maine, USA). Swabs from five birds were pooled together for virus titration and five individual swabs were taken from the remaining birds for virus re-isolation. Individual and pooled swabs were suspended in 2 ml and 4 ml brain–heart infusion broth with antibiotics, respectively, and the suspensions were injected into 10-day-old ECE for virus re-isolation and titration.
Table 2. Replication of AI viruses in experimentally infected chickens
GenBank accession numbers
The nucleotide sequences presented in this article have been submitted to GenBank under the accession numbers AY296064 to AY296086 for the HA, AY300926 to AY300951 for the NA, AY300951 to AY300975 for the MA, and AY300976 to AY300999 for the NS genes.
Results
Isolation of viruses
Virus isolations have been made from three different sources in the US in the past 5 years.
| 1. | H5 subtype viruses have been isolated from domestic ducks, game birds, avian of unspecified species and environmental samples directly associated with the LBMs. Other isolations have been made from pheasants and ducks as part of the surveillance programs required for birds to be sold in the LBMs in the state of New Jersey and New York. | ||||
| 2. | The Texas H5N3 outbreak occurred at a farm owned by a semi-retired egg producer who kept a few chickens (White Leghorns) and leased some of the houses to two LBM operators. The LBM operators used the houses to hold spent brown layers and spent broiler breeders. In April 2002, clinical signs, such as wheezing and swollen heads, were observed in the White Leghorns and 16 out of 111 chickens were dead. Several H5N3 viruses were isolated from those chickens and also from brown layers that did not show clinical signs of disease. There was serological evidence of H5N3 infection in a commercial turkey farm in Texas and in two premises with ducks destined for the LBMs in 1999. | ||||
| 3. | Isolations of H5N2 AI viruses from turkeys were made from frozen lung samples from breeder birds in California. The complex had five houses that were occupied by turkeys from 34 to 55 weeks of age. Flocks had already been determined to be seropositive for AI virus. Infected flocks showed a slight decrease in egg production without clinical signs. | ||||
Pathogenicity of virus isolates
Some of the isolates examined had previously been pathotyped as LPAI viruses (Panigrahy et al., Citation2002). The chickens inoculated intravenously in this study with representative H5 isolates remained clinically healthy during the 10-day observation period. The potential pathogenicity of two H5N2 and the H5N3 isolates were further assessed using a 14-day-old ECE passage system. No HP derivatives were obtained from these viruses.
Sequence and phylogenetic analysis of H5
The coding sequence for the HA or HA1 gene segments from 23 H5 isolates were determined and compared with other sequences available in the sequence database. From the Texas H5N3 outbreak, several viruses were isolated from different breeds of chickens and the poultry house environment. However, they had almost identical sequences in the four genes analyzed in this study, and the A/CK/TX/167280-4/02 isolate was used as a representative strain. The sequence and phylogenetic analysis demonstrated a close relationship between most of the recent H5 subtype isolates from the LBMs, non-LBM premises, wild bird and commercial poultry farms with the exception of the Texas H5N3 isolates. Overall there was at least 94.6% amino acid sequence identity in the HA gene among all recent non-chicken origin isolates. Compared with other recent H5 isolates, the Texas H5N3 isolate shared the lowest homology with other recent isolates in the HA gene and had a unique HA cleavage site sequence of REKR/G (all the other recent isolates have RETR/G motif). The HA cleavage site sequence of different isolates from North America was compared (). The Texas H5N3 virus shared highest sequence identity with A/Chukkar/MN/14951-7/98 (H5N2) isolate, with 93.8% nucleotide and 93.4% amino acid sequence identity.
Table 3. Comparison of HA cleavage site sequences and presence or absence of carbohydrate at amino acid position (Asn) 11 in North American H5 subtype AI viruses
Phylogenetically, all recent isolates were clearly different from the Pennsylvania/83 and LBM/93 lineage isolates and formed a separate clade (). Within recent H5 isolates, at least two distinct clusters were observed. One cluster (Recent A) of viruses contained six viruses obtained from 2000 to 2002, including a wild bird H5N3 isolate (A/Ruddy Turnstone/NJ/2242/00). Another cluster (Recent B) of viruses contained the remaining isolates assorted into several subgroups that contain viruses isolated in the same year. The Texas H5N3 isolate did not belong to any cluster and had a relatively long branch length.
Figure 1. Phylogenetic tree based on nucleotide sequences of the HA1 gene from H5 isolates from the US and other representative isolates from different countries. The tree was generated by the maximum parsimony method with PAUP4.0b10, and is the result of a heuristic search and 100 bootstrap replicates.
The receptor binding site and potential glycosylation sites of the HA1 protein were analyzed and, with the exception of the sequences for the A/Chukkar/MN/14591-7/98, all sequences examined contained completely conserved sequences in the areas surrounding the proposed receptor-binding site. A single amino acid substitution was observed at position 131 (valine to leucine) for the HA1 sequence of A/Chukkar/MN/14591-7/98. Six different potential glycosylation sites were identified and are presented in . All the recent isolates possessed a potential glycosylation site at position 11, where the loss of a glycosylation site has been associated with a virulence shift in field isolates of some H5 subtypes (Kawaoka et al., Citation1984). The Texas H5N3 isolate and three other isolates possessed all six potential glycosylation sites while other isolates were missing at least one site in positions 165, 236, or 286. Glycosylation at position 158, which is thought to be an adaptation to poultry (Matrosovich et al., Citation1999), was not present in any of the recent H5 isolates or the Pennsylvania/83 lineage virus that had circulated in poultry for an extended period of time (Suarez & Senne Citation2000).
Sequence and phylogenetic analysis of N2, N3 and N8 subtype NA
Phylogenetically, the N2 gene assorted into similar groups as compared with the HA1 gene (). One exception was the California turkey isolate, which had a unique N2 sequence and did not cluster with any other recent isolates. The N2 sequences were also not close to the H7N2 subtype isolates from the LBM (Spackman et al., Citation2003) or from the H6N2 subtype isolates from the California outbreak in 2000 to 2001 (Webby et al., Citation2002) ().
Figure 2. Phylogenetic tree based on nucleotide sequences of the N2 gene from H5 isolates from the US and consensus sequences from recent California H6 and LBM H7 subtype isolates. The tree was generated by the maximum parsimony method with PAUP4.0b10, and is the result of a heuristic search and 100 bootstrap replicates.
The N3 gene from the Texas H5N3 isolate was compared with 18 other N3 sequences available with different H subtypes (data not shown). These include a wild bird virus with the same H5N3 subtype (A/Ruddy Turnstone/NJ/2242/00) and a H7N3 wild bird virus from Texas in 2001 (A/Blue-winged teal/TX/2/01) (). No isolates were closely related to the Texas H5N3 isolate. Furthermore, the NA gene of the Texas H5N3 isolate had a 28 amino acid deletion in the stalk region that is not found in other wild bird isolates. One turkey isolate, A/TK/CO/13556/91 (H7N3), has previously been sequenced and had a 26 amino acid deletion in the stalk. We also sequenced a N3 gene from an H7N3 virus (A/CK/NJ/15086-3/94) that is known to be similar to the progenitor strain of H7 subtype virus persisting in the Northeast LBMs (Suarez et al., Citation1999). This isolate had a 24 amino acid stalk deletion. However, the position of the deletion was different among these three isolates and the Texas H5N3 isolate had a one amino acid insertion in the upper part of the stalk region ().
Figure 3. Amino acid sequences comparison of the stalk region of N3. Identical sequences are denoted by dots, and gaps are indicated by dashes. The amino acid numbering is based on the consensus N3 sequence. The threonine insertion in the Texas H5N3 isolate between positions 76 and 77 is indicated by underlining.
One H5 duck isolate from the LBM had an N8 subtype, but few other N8 sequences were available to make meaningful observations from the phylogenetic trees.
Sequence and phylogenetic analysis of the internal proteins
The phylogenetic tree based on the NS gene readily identifies the two previously described subtypes (A and B) of the NS gene (). This separation of recent H5 viruses into subtype A and subtype B is similar to that seen in the tree created from the HA data. All the isolates grouped as Recent As belong to subtype A and Recent Bs belong to subtype B. All recent isolates had a predicted NS1 protein of 230 amino acids and none had deletions that were previously observed with some of the Pennsylvania/83 lineage isolates (Suarez & Perdue, Citation1998).
Figure 4. Phylogenetic tree based on the nucleotide sequences of the NS gene from H5 isolates from the US. The tree was generated by the maximum parsimony method with PAUP4.0b10, and is the result of a heuristic search and midpoint rooting.
In the phylogenetic tree based on the nucleotide sequence of the M gene, several isolates assorted into different groups as compared with those seen in the tree based on HA, NA, and NS genes (). Although this grouping disappeared in a tree based on amino acid sequence, because of the high sequence similarity among recent isolates (data not shown) the tree topology of the M gene based on nucleotide sequence indicates that re-assortment may have occurred. Random distribution of nucleotide substitutions throughout the gene further supports this idea.
Figure 5. Phylogenetic tree based on nucleotide sequences of the M gene from H5 isolates from the US. The tree was generated by the maximum parsimony method with PAUP4.0b10, and is the result of a heuristic search and 100 bootstrap replicates. Isolates assorting into different group than those seen in the trees based on HA, NA, and NS genes are boxed.
Replication of virus in experimentally infected chickens
We compared replication competency of four H5 and one H7 viruses in chickens (). Because only the Texas H5N3 virus was isolated from chickens, we also included a LBM/93 isolate (A/CK/PA/13609/93 (H5N2)) (Horimoto & Kawaoka, Citation1995) and one of the recent H7N2 subtype virus from the LBM (A/CK/NJ/118878-5/01) (Spackman et al., Citation2003). These viruses also show a stalk deletion in the N2 protein. Three-week-old SPF chickens were inoculated intranasally with 0.2 ml allantoic fluid (105.0 EID50/0.2 ml). The birds infected with turkey and duck isolates showed no clinical signs during the observation period. However, birds infected with the three chicken isolates were depressed at 3 d.p.i. Two birds inoculated with the Texas H5N3 isolate showed severe depression at 3 d.p.i. with decreased activity and excessive lacrimation, and eventually died at 6 d.p.i.
At 3 d.p.i., a high titer of virus was isolated from the trachea of all the birds infected with the three chicken isolates (). In contrast, only one bird out of five was positive by virus isolation following inoculation with the California turkey virus (H5N2) and the duck-origin virus (H5N2), respectively. From cloacal swabs, two isolations were made at 3 d.p.i. following inoculation with the duck virus. At 7 d.p.i., the titer of virus in the trachea decreased considerably and the number of virus-positive birds also decreased compared with 3 d.p.i. Only the H7 virus-infected chickens showed a relatively high titer in the trachea, and all five birds were positive. From cloacal swabs, only the H7 virus-infected chickens showed a measurable titer.
Discussion
A study of AI virus subtypes in LBMs and non-LBM premises shows the persistence of the H5 subtype despite efforts to control the infections (Panigrahy et al., Citation2002). This is of concern because certain lineages of this subtype have caused HPAI outbreaks in the US (Eckroade & Bachin, Citation1987). Based on sequencing and animal experiment results, all recent H5N2 viruses from the LBMs and non-LBMs were classified as LPAI virus. Unlike the other recent H5 subtype viruses, the Texas H5N3 virus had an additional basic amino acid at the cleavage site (R-E-K-R). Based on previous observations, this HA cleavage site sequence has the potential to be a virulent motif if the carbohydrate residue near the cleavage site at amino acid position 11 (Asn 11) was removed (Kawaoka et al., Citation1984). Thus, a single point mutation resulting in the removal of this glycosylation site could potentially yield a HP virus. Since this HA cleavage site sequence was unusual, we passaged the virus in 14-day-old ECE, a procedure known to favor the emergence of HP derivatives. Previously, HP virus was derived from a LBM/93 isolate that had a cleavage site sequence of RKTR/G (Horimoto & Kawaoka, Citation1995). No HP derivatives were produced with the Texas H5N3 virus, which may indicate a lower potential for this isolate to become HP. However, the selection pressure in the field is different from that in the laboratory system, which makes interpretation of results difficult.
On the phylogenetic analysis, the recent H5 viruses formed a different clade from the Pennsylvania/83 lineage and also from the LBM/93 lineage viruses. Although the isolates shared a high sequence similarity in the HA protein, genetic evidence shows that recent H5 isolates are likely to be a result of separate introductions from wild birds rather than endemic infection in poultry. Within the recent H5 clade, at least two different subgroups were apparent. Existence of two different subtypes of the NS gene with the same clustering of isolates as the HA gene further reinforces this grouping. Viruses isolated in the same year tend to cluster together in subgroups. No chronological assortment or established lineage was found. This result is in contrast to the well-established Pennsylvania/83 lineage of viruses and recent H7N2 lineages in the US that show a progressive and adaptive evolution in poultry (Suarez et al., Citation1999; Suarez & Senne, Citation2000; Spackman et al., Citation2003).
As with other recent H5 isolates, the Texas H5N3 virus, the only recent chicken-origin isolate in this study, also seems to be a separate introduction from wild waterfowl. However, the evidence suggests that it has been circulating in poultry associated with the LBM for an extended period of time before it was first isolated. First, there were many amino acid changes in the HA gene, including the unique cleavage site sequence, which may indicate an adaptive change in an aberrant host, namely the chicken. Second, the Texas H5N3 isolate had a 28 amino acid deletion in the stalk region of the NA gene, a characteristic of other chicken-adapted influenza viruses, which was not present in any of the other recent H5 isolates. It is known that deletion in the NA gene decreases the ability of the virus to be released from host cells. To compensate for the decreased NA activity for efficient replication of the virus, affinity of the HA for the cell receptor can also be decreased. This can be achieved by acquiring additional carbohydrates at the HA head, which can decrease the affinity of the receptor binding site in the HA to the cell receptor by steric hindrance (Klenk et al., Citation2002). Based on sequence analysis, six different potential glycosylation sites were found in the HA1 subunit of recent H5 isolates. The Texas H5N3 isolate possessed all six potential glycosylation sites while the majority of isolates were missing at least one glycosylation site at position 165 or 236 (). Previously, acquisition of a glycosylation at site 236 was suggested as an adaptive feature of H5 viruses associated with efficient replication in commercial chickens (Garcia et al., Citation1997). However, the presence of both glycosylation sites in the wild bird isolate (A/Ruddy Turnstone/NJ/2242/00) and the relatively distant location of this site from the receptor binding site complicate interpretation of glycosylation at this site. Third, in agreement with genetic evidence, the Texas H5N3 isolate replicated efficiently in chickens and produced clinical signs and even mortality. One interesting observation is that even though the Texas H5N3 and A/CK/PA/13609/93 (H5N2) isolates replicated well in the trachea, the two H5 viruses were cleared from infected birds faster than was the LBM H7N2 virus. Furthermore, the presence of the H7 virus in cloacal swabs may explain why the H7N2 LBM lineage has remained endemic in the LBM since 1994. Fourth, in addition to genetic and virological evidence, previous serological evidence of a positive H5N3 infection of turkey and duck premises in Texas further reinforces the potential circulation of this virus lineage in poultry before its initial isolation in 2002.
In conclusion, the majority of recent H5 isolates were mildly pathogenic and represented typical avirulent waterfowl-like HA cleavage site sequences. However, the outbreak in Texas, with a more chicken-adapted H5N3 virus, and the relationship between this poultry farm and LBM underscores the importance of ongoing surveillance and efforts to control the H5 subtype AI virus in US.
Acknowledgements
The authors thank Suzanne Deblois, Joan Beck, and the SAA sequencing facility for technical assistance with this work. This work was supported by USDA/ARS CRIS project 6612-32000-039.
Related Research Data
References
- Bosch, FX , Orlich, M , Klenk, HD , and Rott, R , 1979. The structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses , Virology 95 (1979), pp. 197–207.
- Brugh, M , and Beck, JR , 1992. "Recovery of minority subpopulation of highly pathogenic avian influenza virus". In: Proceedings of the Third International Symposium on Avian Influenza, University of Wisconsin . Madison, WI. 1992. p. (pp. 166–174), In B.C. Easterday (Ed.),.
- Eckroade, RJ , and Bachin, LA , 1987. "Avian influenza in Pennsylvania—the beginning". In: Proceedings of the Second International Symposium on Avian Influenza, Athens, GA . 1987. p. (pp. 22–32), In C.W. Beard & B.C. Easterday (Eds.),.
- Garcia, M , Crawford, JM , Latimer, JW , Rivera-Cruz, E , and Perdue, ML , 1996. Heterogeneity in the haemagglutinin gene and emergence of the highly pathogenic phenotype among recent H5N2 avian influenza viruses from Mexico , Journal of General Virolology 77 (1996), pp. 1493–1504.
- Garcia, M , Suarez, DL , Crawford, JM , Latimer, JW , Slemons, RD , Swayne, DE , and Perdue, ML , 1997. Evolution of H5 subtype avian influenza A viruses in North America , Virus Research 51 (1997), pp. 115–124.
- Horimoto, T , and Kawaoka, Y , 1995. Molecular changes in virulent mutants arising from avirulent avian influenza viruses during replication in 14-day-old embryonated eggs , Virology 206 (1995), pp. 755–759.
- Kawaoka, Y , Naeve, CW , and Webster, RG , 1984. Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? , Virology 139 (1984), pp. 303–316.
- Klenk, H-D , Wagner, R , Heuer, D , and Wolff, T , 2002. Importance of hemagglutinin glycosylation for the biological functions of influenza virus , Virus Research 82 (2002), pp. 73–75.
- Lang, G , Marayan, O , Rouse, BT , Fergispm, AE , and Connell, MC , 1968. A new influenza virus A infection in turkeys. II. A highly pathogenic variant, A/turkey/Ontario/7732/66 , Canadian Veterinary Journal 9 (1968), pp. 151–160.
- 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 (1999), pp. 1146–1155.
- Mitnaul, LJ , Matrosovich, MN , Castrucci, MR , Tuzikov, AB , Bovin, NV , Kobasa, D , and Kawaoka, Y , 2000. Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza a virus , Journal of Virology 74 (2000), pp. 6015–6020.
- Panigrahy, B , Senne, DA , and Pedersen, JC , 2002. Avian influenza virus subtypes inside and outside the live bird markets, 1993–2000: a spatial and temporal relationship , Avian Diseases 46 (2002), pp. 298–307.
- Perdue, ML , Garcia, M , Beck, J , Brugh, M , and Swayne, D , 1996. An Arg-Lys insertion at the hemagglutinin cleavage site of an H5N2 avian influenza isolate , Virus Genes 12 (1996), pp. 77–84.
- Senne, DA , Pearson, JE , and Panigrahy, B , 1992. "Live poultry markets: a missing link in the epidemiology of avian influenza". In: Proceedings of the Third International Symposium on Avian Influenza . Madison, WI. 1992. p. (pp. 50–58).
- Senne, DA , Rivera, E , Panigrahy, B , Fraire, M , Kawaoka, Y , and Webster, RG , 1996a. "Characterization of avian influenza H5N2 isolates recovered from chickens in Mexico". In: Proceedings of the 45th Western Poultry Disease Conference . Cancun, Mexico. 1996a. p. (pp. 5–8).
- Senne, DA , Panigrahy, B , Kawaoka, Y , Pearson, JE , Suss, J , Lipkind, M , Kida, H , and Webster, RG , 1996b. 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 Diseases 40 (1996b), pp. 425–437.
- Spackman, E , Senne, DA , Myers, TJ , Bulaga, LL , Garber, LP , Perdue, ML , Lohman, K , Daum, LT , and Suarez, DL , 2002. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes , Journal of Clinical Microbiology 40 (2002), pp. 3256–3260.
- Spackman, E , Senne, DA , Davison, S , and Suarez, DL , 2003. Sequence analysis of recent H7 avian influenza viruses associated with three different outbreaks in commercial poultry in the United States , Journal of Virology 77 (2003), pp. 13399–13402.
- Suarez, DL , and Perdue, ML , 1998. Multiple alignment comparison of the non-structural genes of influenza A viruses , Virus Research 54 (1998), pp. 59–69.
- Suarez, DL , and Senne, DA , 2000. Sequence analysis of related low-pathogenic and highly pathogenic H5N2 avian influenza isolates from United States live bird markets and poultry farms from 1983 to 1989 , Avian Diseases 44 (2000), pp. 356–364.
- Suarez, DL , Perdue, ML , Cox, N , Rowe, T , Bender, C , Huang, J , and Swayne, DE , 1998. Comparisons of highly virulent H5N1 influenza A viruses isolated from humans and chickens from Hong Kong , Journal of Virology 72 (1998), pp. 6678–6688.
- Suarez, DL , Garcia, M , Latimer, J , Senne, D , and Perdue, M , 1999. Phylogenetic analysis of H7 avian influenza viruses isolated from the live bird markets of the Northeast United States , Journal of Virology 73 (1999), pp. 3567–3573.
- Suarez, DL , Senne, DA , Banks, J , Brown, IH , Essen, SC , Lee, CW , Manvell, RJ , Mathieu-Benson, C , Mareno, V , Pedersen, J , Panigrahy, B , Rojas, H , Spackman, E , and Alexander, DJ , 2004. A virulence shift in the influenza A subtype H7N3 virus responsible for a natural outbreak of avian influenza in Chile appears to be the result of recombination , Emerging Infectious Diseases 10 ((4)) (2004).
- Swayne, DE , Beck, JR , Garcia, M , Perdue, ML , and Brugh, M , 1998. "Pathogenicity shifts in experimental avian influenza virus infections in chickens". In: Proceedings of the Fourth International Symposium on Avian Influenza, Richmond, VA . 1998. p. (pp. 171–181), In D.E. Swayne & R.D. Slemons (Eds.),.
- Swofford, DL , 1998. PAUP*. Phylogenetic Analysis using Parsimony (*and Other Methods) . Sunderland, MA. 1998, Version 4..
- 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 (2000), pp. 6316–6323.
- Webby, RJ , Woolcock, PR , Krauss, SL , and Webster, RG , 2002. Reassortment and interspecies transmission of North American H6N2 influenza viruses , Virology 295 (2002), pp. 44–53.