BACKGROUND
avian influenza  virus belongs to the orthomyxoviridae family, influenzavirus a genus.
this virus possesses eight segments of single-stranded rna genome.
two of these segments encode for two important membrane glycoproteins, hemagglutinin  and neuraminidase   <cit> , that play a key role during cellular infection.
these two proteins are used for virus subtype classification  <cit> .
also, depending on severity of disease in avian species, ai viruses are categorized into highly- and low-pathogenic  viruses  <cit> .
in response to the recent cases of human infections caused by hpai h5n <dig> viruses, authorities and scientists were encouraged to review and apply policies for effective surveillance and control of ai infections  <cit> .
in many countries, the use of ai vaccines was banned or discouraged because vaccination programs could interfere with appropriate detection of hpai outbreaks  <cit> .
however, the use of ai vaccines has been reconsidered by some countries due to the recent increase in ai cases in commercial farms and devastating consequences for human health  <cit> .
in mexico, an ai vaccination program was established in  <dig>  initially, the program was instituted to control the hpai h5n <dig> virus outbreak that occurred during that year  <cit> .
a commercial vaccine against ai was produced using the officially authorized virus strain a/ck/méxico/cpa-232/ <dig>  a few months later, the hpai virus was eradicated from mexico and it was decided to continue the vaccination program to protect commercial flocks from lpai h5n <dig> viruses  <cit> .
after almost two decades of using the ai vaccine in mexico, commercial farms remain hpai-free.
however, veterinary services have observed an increase in respiratory signs in vaccinated, field challenged  birds.
moreover, animal health laboratories have reported significant differences in the hemagglutination inhibition  tests between field lpai h5n <dig> isolates and the vaccine strain  <cit> .
these discrepancies observed during ai surveillance could be attributed to a gradual accumulation of antigenic drift.
in fact, it was shown that lpai h5n <dig> viruses in mexico are constantly undergoing genetic drift, and that recent ai virus isolates have significant antigen divergence when compared to the ai vaccine strain  <cit> .
in mexico, as in many other countries, ai surveillance is primarily carried out by the hi test using reference antigens or antisera  <cit> .
this method is recommended by the world organization for animal health  as standard test to detect antigenic differences  between circulating, vaccine and reference ai virus strains  <cit> , and to evaluate vaccine efficacy  <cit> .
antigens for production of vaccines or antisera are maintained and distributed by official reference laboratories  <cit>  and in many cases, these antigens are produced with ai viruses isolated more than a decade ago .
although the hi test and reference antigens are used worldwide for ai surveillance; little has been done to examine the effects of ai antigenic drift on the antigenic surveillance of field strains.
we hypothesize that the antigenic drift that occurred in recent field isolates of h5n <dig> virus produced significant variation in the accuracy of the serologic surveillance.
the objective of the present study was to examine the effects of ai antigenic drift on immune reactivity of reference antisera using standard hi tests.
RESULTS
low pathogenic ai h5n <dig> viruses isolated in mexico between  <dig> and  <dig> were used for phylogenetic analyses of ai hemagglutinin genes and immune reactivity using antisera produced with ai virus isolated in different years.
phylogenetic analysis of avian influenza hemagglutinin genes
field isolates of lpai h5n <dig> virus were used for this study.
these viruses were isolated from vaccinated birds that developed the clinical presentation of the disease.
virus were replicated in chicken embryo and reverse transcriptase pcr was used for the amplification of the ha gene , a marker for the virulence potential of ai viruses  <cit> .
ha gene segments were sequenced and annotated for phylogenetic analyses.
to accomplish a more comprehensive evolutionary history of ai h5n <dig> viruses in mexico, all available a/ck/méxico/h5n <dig> sequences in the influenza virus resource at the national center for biotechnology information  <cit>  were retrieved and used for genetic analysis.
phylogenic analysis of partial ha gene sequences from ai h5n <dig> viruses isolated between  <dig> and  <dig> revealed that the ai viruses are constantly undergoing genetic drifts.
phylogenetic trees derived from partial nucleotide sequences demonstrated that ha genes amplified from field lpai h5n <dig> isolates distinctively cluster by year of isolation .
similar tree topologies were obtained by the maximum likelihood method .
these phylogenetic analysis were confirmed by estimating percent sequence identity scores for pairwise comparisons of lpai h5n <dig> isolates and the vaccine strain .
together, these data indicate that recent lpai h5n <dig> isolates  have undergone significant molecular drifts when compared to the vaccine strain , isolated in  <dig> and early viruses isolated between  <dig> and  <dig> .
this analysis confirms that ha gene lineages in mexico follow a yearly cumulative trend of sequence mutations.
interestingly, one of the most recent virus isolates  isolated in  <dig>  was closely related to the vaccine strain and early virus isolates .
this result could indicate that at some point a vaccine-like virus may have circulated in commercial flocks during  <dig>
percent of sequence similarity between the reference strain  and low pathogenic avian influenza h5n <dig> viruses isolated between  <dig> and  <dig>  mean values ± sd.
statistical comparisons were made using anova and fisher's protected least significant difference test.
values with different superscript are significantly different .
the phylogenetic analysis also revealed significant divergence between different strains of lpai h5n <dig> isolates in mexico.
viruses circulating between  <dig> and  <dig> were highly divergent to viruses isolated between  <dig> and  <dig> .
these results indicate that current ai virus circulating in mexico possesses sufficient genetic divergence to warrant a vaccine strain update.
to assess the effect of this antigenic divergence between field isolates and the official vaccine strain, serologic testing was evaluated by means of hi assays.
serologic testing using hemagglutination inhibition tests
the thirty-four field isolates of lpai h5n <dig> virus were used to evaluate immune reactivity using standard hi tests.
ai antisera was produced using seven year-specific ai h5n <dig> antigens: a) a/ck/méxico/cpa-232/ <dig>  reference antigen and virus strain used for vaccine production; b) a/ck/méxico/2002; c) a/ck/méxico/2003; d) a/ck/méxico/2005; e) a/ck/méxico/2006; f) a/ck/méxico/ <dig> and g) a/ck/méxico/ <dig>  the hi assay demonstrated highly variable results when field lpai h5n <dig> viruses where tested against antisera derived from year-specific ai h5n <dig> strains.
in the hi tests using antiserum produced with antigen a/ck/méxico/cpa-232/ <dig>  geometric mean titers  were significantly higher in lpai h5n <dig> viruses isolated in  <dig> compared to viruses isolated between  <dig>   <dig> and  <dig> .
the hi test gmt of viruses isolated between  <dig> and  <dig> were comparable.
these results indicate that at the ha antigenic level,  <dig> and  <dig> lpai viruses were more closely related to a/ck/méxico/cpa-232/ <dig> than more recent virus isolates  .
antigenic relatedness of the reference strain  and low pathogenic avian influenza h5n <dig> viruses isolated between  <dig> and  <dig> tested by hemagglutination inhibition  test.
official virus id: a).
a/ck/méxico/1994; b).
a/ck/méxico/2002; c).
a/ck/méxico/2003; d).
a/ck/méxico/2005; e).
a/ck/méxico/2006; f).
a/ck/méxico/2007; g).
a/ck/méxico/ <dig>  geometric mean values for hi test using different antisera  and low pathogenic avian influenza h5n <dig> viruses isolated between  <dig> and  <dig>  statistical comparisons were made using anova and fisher's protected least significant difference test.
columns with different superscript are significantly different .
for the hi test using antiserum produced with antigen a/ck/méxico/ <dig>  gmt revealed close homology between the  <dig> virus isolates; however, these gmt did not follow a yearly trend for the other years.
for example,  <dig> lpai isolates were antigenically divergent to a/ck/méxico/ <dig> whereas no differences were detected between viruses isolated in  <dig> and  <dig>  in contrast,  <dig> isolates were antigenically divergent when compared to  <dig> viruses; indicating highly antigenic divergence between viruses isolated at close time points .
when a/ck/méxico/ <dig> and a/ck/méxico/ <dig> antisera were used for the hi assays, antigenic homology was comparable between lpai viruses isolated in  <dig>   <dig>   <dig>   <dig> and  <dig>  these results indicate that at the ha antigenic level, this collection of lpai viruses possess comparable antigenic homology with the a/ck/méxico/ <dig> virus .
nevertheless, it is noteworthy that the gmt in the hi test were highly variable.
in the hi test using the a/ck/méxico/ <dig>  lpai viruses isolated between  <dig> and  <dig> showed comparable antigenic homology.
in contrast, viruses isolated during  <dig> showed a reduced antigenic homology when compared to  <dig> lpai isolates.
.
the hi test using antiserum a/ck/méxico/ <dig> followed a yearly trend.
lpai viruses isolated in  <dig> and  <dig> showed comparable antigenic homology.
in contrast, these lpai isolates were antigenically divergent to early isolates  and more recent isolates .
these results indicate the lpai h5n <dig> viruses circulating during  <dig> and  <dig> possess year-specific antigenic divergence compared to other years of isolation.
comparable trend was observed in the hi test using the a/ck/méxico/ <dig> antiserum.
lpai viruses circulating between  <dig> and  <dig> were more antigenically related to the a/ck/méxico/ <dig> virus .
these results corroborate that recent ai viruses have accumulated significant antigenic drifts to be distinguishable from early ai isolates .
taken together, these results show some yearly tendencies in antigenic homology in which early lpai isolated strains  are more antigenically related to the vaccine strain a/ck/méxico/cpa-232/ <dig> and recent lpai isolates  more antigenically related to the a/ck/méxico/ <dig> virus .
these results indicate that the antigenic differences of circulating lpai h5n <dig> viruses may warrant a new vaccine and serosurveillance antigen update.
discussion
worldwide, human and animal health organizations have established detailed schemes for influenza surveillance.
these programs rely on serological assays to characterize virus subtypes, establishing seroprevalence and evaluating vaccine efficacy  <cit> .
in veterinary medicine, the hi test is the standard technique used to detect antigenic differences between circulating influenza virus, vaccine- and/or reference- strains  <cit> .
because the hi test is used worldwide for ai surveillance, it is essential to identify potential pitfalls of using this serologic test.
the objective of the present study was to examine the effects of ai antigenic drift on serosurveillance using reference antisera during standard hi tests.
we amplified, sequenced and analyzed partial ai hemagglutinin genes and performed serologic tests using lpai h5n <dig> viruses isolated in mexico between  <dig> and  <dig>  phylogenetic analyses revealed that molecular drift in ha gene follow a yearly trend, suggesting gradually cumulative sequence mutations.
recent  field isolates of lpai h5n <dig> viruses in mexico have undergone important antigenic drift in the ha gene when compared to early lpai isolates or vaccine strains .
viruses isolated between  <dig> and  <dig> cluster in distinctive and divergent lineages compared to viruses isolated between  <dig> and  <dig>  comparable evolutionary trends were observed in previous analyses of lpai h5n <dig> lineages in mexico  <cit> .
these evolving genetic drifts observed in ha genes of lpai h5n <dig> may explain the increasing incidence of respiratory signs in vaccinated, field challenged birds, and the discrepancies observed during lpai h5n <dig> serosurveillance.
to confirm this idea, serologic testing was carried out using an array of seven different reference ai antisera produced with lpai h5n <dig> antigens isolated in  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  these analyses revealed that hi test results are significantly influenced by the selection of year-specific antigens.
generally, early lpai h5n <dig> virus isolates  produced higher hi titers with antisera produced with early isolates .
in contrast, more recent lpai h5n <dig> isolates  produced higher hi titers with antisera produced with recent lpai isolated strains .
these results explain the high variability of lpai titers in commercial flocks when the reference ai antigen a/ck/méxico/cpa-232/ <dig> is used in the hi test.
despite that considerable efforts have been made to produce reference antibodies to improve the accuracy of the hi test during serosurveillance of ai  <cit> , the accelerated mutation rate of ai viruses and the rapid accumulation of antigen drift  <cit>  require a recurrent update of reference antigens or antisera for accurate surveillance.
for example, in mexico around  <dig>  veterinary services reported that ai serum titers in commercial flocks were highly variable when the reference antigen a/ck/méxico/cpa-232/ <dig>  was used for the hi test.
similar discrepancies between reference antisera and field isolates were observed in the serosurveillance of h3n <dig> and h1n <dig> influenza a viruses in pigs  <cit> .
our results confirm the idea that the accelerated antigenic drift observed in ai viruses, not only affects the performance and accuracy of the hi test during ai serosurveillance but also, the effectiveness of ai vaccines  <cit> .
hence, for an effective control of ai h5n <dig> viruses, reference antigens and vaccine strains must be replaced constantly to keep pace with the ai antigenic drift.
this approach has been a successful strategy for the control and surveillance of human influenza  <cit> .
CONCLUSIONS
the present study reveals that due to the rapid antigenic drift of ai viruses, standardization and constant renewal of reference antigens is required during the establishment of influenza serosurveillance programs.
more importantly, these data provide clear evidence of the impact of antigenic drifts on the evasion of the immune system.
antibodies produced against early ai viruses  have reduced effectiveness against lpai viruses currently circulating in the environment.
thus, the observed genetic and antigenic differences of circulating lpai h5n <dig> viruses warrant an update of the vaccine strain and serosurveillance antigens.

 avian influenza virus belongs to the orthomyxoviridae family, influenzavirus a genus. this virus possesses eight segments of single-stranded rna genome. two of these segments encode for two important membrane glycoproteins, hemagglutinin and neuraminidase, that play a key role during cellular infection. these two proteins are used for virus subtype classification. also, depending on severity of disease in avian species, ai viruses are categorized into highly- and low-pathogenic viruses. in response to the recent cases of human infections caused by hpai h5n viruses, authorities and scientists were encouraged to review and apply policies for effective surveillance and control of ai infections. in many countries, the use of ai vaccines was banned or discouraged because vaccination programs could interfere with appropriate detection of hpai outbreaks. however, the use of ai vaccines has been reconsidered by some countries due to the recent increase in ai cases in commercial farms and devastating consequences for human health. in mexico, an ai vaccination program was established in initially, the program was instituted to control the hpai h5n virus outbreak that occurred during that year. a commercial vaccine against ai was produced using the officially authorized virus strain a/ck/méxico/cpa-232/ a few months later, the hpai virus was eradicated from mexico and it was decided to continue the vaccination program to protect commercial flocks from lpai h5n viruses. after almost two decades of using the ai vaccine in mexico, commercial farms remain hpai-free. however, veterinary services have observed an increase in respiratory signs in vaccinated, field challenged birds. moreover, animal health laboratories have reported significant differences in the hemagglutination inhibition tests between field lpai h5n isolates and the vaccine strain. these discrepancies observed during ai surveillance could be attributed to a gradual accumulation of antigenic drift. in fact, it was shown that lpai h5n viruses in mexico are constantly undergoing genetic drift, and that recent ai virus isolates have significant antigen divergence when compared to the ai vaccine strain. in mexico, as in many other countries, ai surveillance is primarily carried out by the hi test using reference antigens or antisera. this method is recommended by the world organization for animal health as standard test to detect antigenic differences between circulating, vaccine and reference ai virus strains, and to evaluate vaccine efficacy. antigens for production of vaccines or antisera are maintained and distributed by official reference laboratories and in many cases, these antigens are produced with ai viruses isolated more than a decade ago. although the hi test and reference antigens are used worldwide for ai surveillance; little has been done to examine the effects of ai antigenic drift on the antigenic surveillance of field strains. we hypothesize that the antigenic drift that occurred in recent field isolates of h5n virus produced significant variation in the accuracy of the serologic surveillance. the objective of the present study was to examine the effects of ai antigenic drift on immune reactivity of reference antisera using standard hi tests.  low pathogenic ai h5n viruses isolated in mexico between and were used for phylogenetic analyses of ai hemagglutinin genes and immune reactivity using antisera produced with ai virus isolated in different years. phylogenetic analysis of avian influenza hemagglutinin genes field isolates of lpai h5n virus were used for this study. these viruses were isolated from vaccinated birds that developed the clinical presentation of the disease. virus were replicated in chicken embryo and reverse transcriptase pcr was used for the amplification of the ha gene, a marker for the virulence potential of ai viruses. ha gene segments were sequenced and annotated for phylogenetic analyses. to accomplish a more comprehensive evolutionary history of ai h5n viruses in mexico, all available a/ck/méxico/h5n sequences in the influenza virus resource at the national center for biotechnology information were retrieved and used for genetic analysis. phylogenic analysis of partial ha gene sequences from ai h5n viruses isolated between and revealed that the ai viruses are constantly undergoing genetic drifts. phylogenetic trees derived from partial nucleotide sequences demonstrated that ha genes amplified from field lpai h5n isolates distinctively cluster by year of isolation. similar tree topologies were obtained by the maximum likelihood method. these phylogenetic analysis were confirmed by estimating percent sequence identity scores for pairwise comparisons of lpai h5n isolates and the vaccine strain. together, these data indicate that recent lpai h5n isolates have undergone significant molecular drifts when compared to the vaccine strain, isolated in and early viruses isolated between and. this analysis confirms that ha gene lineages in mexico follow a yearly cumulative trend of sequence mutations. interestingly, one of the most recent virus isolates isolated in was closely related to the vaccine strain and early virus isolates. this result could indicate that at some point a vaccine-like virus may have circulated in commercial flocks during percent of sequence similarity between the reference strain and low pathogenic avian influenza h5n viruses isolated between and mean values ± sd. statistical comparisons were made using anova and fisher's protected least significant difference test. values with different superscript are significantly different. the phylogenetic analysis also revealed significant divergence between different strains of lpai h5n isolates in mexico. viruses circulating between and were highly divergent to viruses isolated between and. these results indicate that current ai virus circulating in mexico possesses sufficient genetic divergence to warrant a vaccine strain update. to assess the effect of this antigenic divergence between field isolates and the official vaccine strain, serologic testing was evaluated by means of hi assays. serologic testing using hemagglutination inhibition tests the thirty-four field isolates of lpai h5n virus were used to evaluate immune reactivity using standard hi tests. ai antisera was produced using seven year-specific ai h5n antigens: a) a/ck/méxico/cpa-232/ reference antigen and virus strain used for vaccine production; b) a/ck/méxico/2002; c) a/ck/méxico/2003; d) a/ck/méxico/2005; e) a/ck/méxico/2006; f) a/ck/méxico/ and g) a/ck/méxico/ the hi assay demonstrated highly variable results when field lpai h5n viruses where tested against antisera derived from year-specific ai h5n strains. in the hi tests using antiserum produced with antigen a/ck/méxico/cpa-232/ geometric mean titers were significantly higher in lpai h5n viruses isolated in compared to viruses isolated between and. the hi test gmt of viruses isolated between and were comparable. these results indicate that at the ha antigenic level, and lpai viruses were more closely related to a/ck/méxico/cpa-232/ than more recent virus isolates. antigenic relatedness of the reference strain and low pathogenic avian influenza h5n viruses isolated between and tested by hemagglutination inhibition test. official virus id: a). a/ck/méxico/1994; b). a/ck/méxico/2002; c). a/ck/méxico/2003; d). a/ck/méxico/2005; e). a/ck/méxico/2006; f). a/ck/méxico/2007; g). a/ck/méxico/ geometric mean values for hi test using different antisera and low pathogenic avian influenza h5n viruses isolated between and statistical comparisons were made using anova and fisher's protected least significant difference test. columns with different superscript are significantly different. for the hi test using antiserum produced with antigen a/ck/méxico/ gmt revealed close homology between the virus isolates; however, these gmt did not follow a yearly trend for the other years. for example, lpai isolates were antigenically divergent to a/ck/méxico/ whereas no differences were detected between viruses isolated in and in contrast, isolates were antigenically divergent when compared to viruses; indicating highly antigenic divergence between viruses isolated at close time points. when a/ck/méxico/ and a/ck/méxico/ antisera were used for the hi assays, antigenic homology was comparable between lpai viruses isolated in and these results indicate that at the ha antigenic level, this collection of lpai viruses possess comparable antigenic homology with the a/ck/méxico/ virus. nevertheless, it is noteworthy that the gmt in the hi test were highly variable. in the hi test using the a/ck/méxico/ lpai viruses isolated between and showed comparable antigenic homology. in contrast, viruses isolated during showed a reduced antigenic homology when compared to lpai isolates.. the hi test using antiserum a/ck/méxico/ followed a yearly trend. lpai viruses isolated in and showed comparable antigenic homology. in contrast, these lpai isolates were antigenically divergent to early isolates and more recent isolates. these results indicate the lpai h5n viruses circulating during and possess year-specific antigenic divergence compared to other years of isolation. comparable trend was observed in the hi test using the a/ck/méxico/ antiserum. lpai viruses circulating between and were more antigenically related to the a/ck/méxico/ virus. these results corroborate that recent ai viruses have accumulated significant antigenic drifts to be distinguishable from early ai isolates. taken together, these results show some yearly tendencies in antigenic homology in which early lpai isolated strains are more antigenically related to the vaccine strain a/ck/méxico/cpa-232/ and recent lpai isolates more antigenically related to the a/ck/méxico/ virus. these results indicate that the antigenic differences of circulating lpai h5n viruses may warrant a new vaccine and serosurveillance antigen update. discussion worldwide, human and animal health organizations have established detailed schemes for influenza surveillance. these programs rely on serological assays to characterize virus subtypes, establishing seroprevalence and evaluating vaccine efficacy. in veterinary medicine, the hi test is the standard technique used to detect antigenic differences between circulating influenza virus, vaccine- and/or reference- strains. because the hi test is used worldwide for ai surveillance, it is essential to identify potential pitfalls of using this serologic test. the objective of the present study was to examine the effects of ai antigenic drift on serosurveillance using reference antisera during standard hi tests. we amplified, sequenced and analyzed partial ai hemagglutinin genes and performed serologic tests using lpai h5n viruses isolated in mexico between and phylogenetic analyses revealed that molecular drift in ha gene follow a yearly trend, suggesting gradually cumulative sequence mutations. recent field isolates of lpai h5n viruses in mexico have undergone important antigenic drift in the ha gene when compared to early lpai isolates or vaccine strains. viruses isolated between and cluster in distinctive and divergent lineages compared to viruses isolated between and comparable evolutionary trends were observed in previous analyses of lpai h5n lineages in mexico. these evolving genetic drifts observed in ha genes of lpai h5n may explain the increasing incidence of respiratory signs in vaccinated, field challenged birds, and the discrepancies observed during lpai h5n serosurveillance. to confirm this idea, serologic testing was carried out using an array of seven different reference ai antisera produced with lpai h5n antigens isolated in and these analyses revealed that hi test results are significantly influenced by the selection of year-specific antigens. generally, early lpai h5n virus isolates produced higher hi titers with antisera produced with early isolates. in contrast, more recent lpai h5n isolates produced higher hi titers with antisera produced with recent lpai isolated strains. these results explain the high variability of lpai titers in commercial flocks when the reference ai antigen a/ck/méxico/cpa-232/ is used in the hi test. despite that considerable efforts have been made to produce reference antibodies to improve the accuracy of the hi test during serosurveillance of ai, the accelerated mutation rate of ai viruses and the rapid accumulation of antigen drift require a recurrent update of reference antigens or antisera for accurate surveillance. for example, in mexico around veterinary services reported that ai serum titers in commercial flocks were highly variable when the reference antigen a/ck/méxico/cpa-232/ was used for the hi test. similar discrepancies between reference antisera and field isolates were observed in the serosurveillance of h3n and h1n influenza a viruses in pigs. our results confirm the idea that the accelerated antigenic drift observed in ai viruses, not only affects the performance and accuracy of the hi test during ai serosurveillance but also, the effectiveness of ai vaccines. hence, for an effective control of ai h5n viruses, reference antigens and vaccine strains must be replaced constantly to keep pace with the ai antigenic drift. this approach has been a successful strategy for the control and surveillance of human influenza.  the present study reveals that due to the rapid antigenic drift of ai viruses, standardization and constant renewal of reference antigens is required during the establishment of influenza serosurveillance programs. more importantly, these data provide clear evidence of the impact of antigenic drifts on the evasion of the immune system. antibodies produced against early ai viruses have reduced effectiveness against lpai viruses currently circulating in the environment. thus, the observed genetic and antigenic differences of circulating lpai h5n viruses warrant an update of the vaccine strain and serosurveillance antigens. 
however, significant discrepancies between reference antisera and field isolates have been observed during serosurveillance of influenza a viruses in pig and poultry farms. phylogenetic analysis revealed significant divergence between early lpai h5n viruses and more recent virus field isolates. serologic surveillance of avian influenza viruses is carried out by the hemagglutination inhibition test using reference reagents. results of the hi test were markedly influenced by the selection of the ai h5n virus used as reference antigen for the assay. reference viral antigens and/or antisera need to be replaced constantly during surveillance of ai viruses to keep pace with the ai antigenic drift. the objective of this study was to examine the effects of influenza virus genetic and antigenic drift on serologic testing using standard hi assays and reference reagents. this method is recommended by animal health organizations as a standard test to detect antigenic differences between circulating influenza virus, vaccine- and/or reference- strains. these analyses indicate that lpai h5n viruses in mexico are constantly undergoing genetic drift and that serosurveillance of ai viruses is significantly influenced by the antigen or antisera used for the hi test. low pathogenic ai h5n viruses isolated in mexico between and were used for phylogenetic analysis of ai hemagglutinin genes and for serologic testing using antisera produced with year-specific ai virus isolates. this strategy should improve the estimation of antigenic differences between circulating ai viruses and the selection of suitable vaccine strains.
 serologic surveillance of avian influenza viruses is carried out by the hemagglutination inhibition test using reference reagents. this method is recommended by animal health organizations as a standard test to detect antigenic differences between circulating influenza virus, vaccine- and/or reference- strains. however, significant discrepancies between reference antisera and field isolates have been observed during serosurveillance of influenza a viruses in pig and poultry farms. the objective of this study was to examine the effects of influenza virus genetic and antigenic drift on serologic testing using standard hi assays and reference reagents. low pathogenic ai h5n viruses isolated in mexico between and were used for phylogenetic analysis of ai hemagglutinin genes and for serologic testing using antisera produced with year-specific ai virus isolates.  phylogenetic analysis revealed significant divergence between early lpai h5n viruses and more recent virus field isolates. results of the hi test were markedly influenced by the selection of the ai h5n virus used as reference antigen for the assay. these analyses indicate that lpai h5n viruses in mexico are constantly undergoing genetic drift and that serosurveillance of ai viruses is significantly influenced by the antigen or antisera used for the hi test.  reference viral antigens and/or antisera need to be replaced constantly during surveillance of ai viruses to keep pace with the ai antigenic drift. this strategy should improve the estimation of antigenic differences between circulating ai viruses and the selection of suitable vaccine strains. 