BACKGROUND
avian influenza virus  is a type a virus of the family orthomyxovirida.
although wild aquatic birds such as water fowls and sea gulls are their natural reservoir  <cit> , land-based birds including chickens may also be infected, which cause significant economic losses to the poultry industry, and raise a great public health threat due to potential host jump from animals to humans  <cit> .
mirnas are non-coding, single-stranded rnas of 19~ <dig> nucleotides which represent a novel class of gene regulators and play important roles in a variety of biological processes in both plants and animals  <cit> .
mirnas modulate gene expression largely at the post-transcriptional level by different mechanisms including direct cleavage of targeted mrnas  <cit> , inhibition of translation  <cit>  or even up-regulation of translation  <cit> .
mirnas are involved in different biological activities such as development, differentiation, growth and metabolism  <cit> .
recently, in mammals, mirnas have been reported to participate in the regulation of immunity, including development and differentiation of lymphocytes, monocytes and neutrophils, and modulation of inflammation  <cit> .
mir- <dig> expresses in mature b and t cells derived from mouse hematopoietic stem cells, and is able to block early b cell development when expressed prematurely  <cit> .
mir-181a is an intrinsic modulator of t cell sensitivity and selection in mice  <cit> .
after exposure of thp- <dig>  cells to lipopolysaccharides , mir- <dig> was identified as an inhibitor of signalling proteins of the innate immune responses by nf-kappab  <cit> .
mirnas have also been found to be critical effectors in the regulation of viral pathogenesis.
two human encoded mirnas , have been shown to have potential binding sites for the genes that code for the polymerase basic  <dig>  and hemmagglutinin  proteins and are reported to be involved in the pathogenesis of h5n <dig> aiv  <cit> .
all of these evidences suggest that certain mirnas might be important in the modulation of aiv infections in chickens.
in order to effectively control aiv infection in poultry, it is essential to elucidate the mechanisms of virus pathogenesis in chickens.
however, how host cells interact with aivs during infection in poultry remains poorly understood.
identification of differentially expressed mirnas in aiv infected chickens will pave a novel avenue to understand host-virus interaction.
with the development of next generation sequencing, massively parallel sequencing holds great promise for expression profiling  <cit>  and it can provide a superior sensitivity at high sequencing depth to discover especially those mirnas with low abundance and novel mirnas that are not able to be identified using traditional cloning approaches.
deep sequencing has been previously used to profile both chicken mirnas and marek's disease virus mirnas  <cit> .
in the current study, a solexa sequencer was used to deep sequence differentially regulated chicken mirnas in h5n <dig> infected and non-infected spf chickens.
our results will expand the list of mirnas which might be related to the host immune responses in animals.
RESULTS
virus titration
virus replication was examined by real-time rt-pcr for influenza matrix gene from total rnas of lung and tracheae at  <dig> dpi.
the titer of infected samples was  <dig>  log <dig> eid50/ml in lung, and  <dig>  log <dig> eid50/ml in tracheae.
both non-infected lung and tracheae samples were negative.
small rna libraries
a total of  <dig>  and  <dig>  filtered high quality reads were obtained from chicken lungs and tracheae, respectively .
in the libraries of chicken lungs,  <dig>  and  <dig>  reads were obtained from infected and non-infected lungs, respectively.
out of these reads,  <dig>  of these high quality reads were exact matches while another  <dig>  reads were loose matches to known chicken mirnas.
all reads with a perfect match to mature mirna sequences from chicken deposited in mirbase  with insertions or deletions of 1- <dig> nucleotides was considered as a loose match to represent dicer-processing products from each of the chicken mirna precursors.
an example from gga-mir-181a is shown in figure  <dig>  here we saw that the sample had  <dig> copies of a sequence that was identical to that of the mature gga-mir-181a .
in addition we observed significant alternate processing at the 3'-end that was characteristic of mirnas in various copy numbers.
loose matches were defined by sequence reads that aligned with chicken mirna consensus sequence with 1- <dig> mismatches.
these may represent sequencing errors , mutations and/or rna editing events.
in the libraries of chicken trachea,  <dig>  reads were obtained from infected tracheae and  <dig>  were obtained from non-infected tracheae.
out of these reads,  <dig>  of these high quality reads were exact matches and another  <dig>  reads were loose matches to known chicken mirnas.
the sum of exact and loose match reads was used as the total number of reads for each mirna.
to display the distribution of mirnas at each library, reads of individual mirna within each group were transformed to log <dig>  the plots of distributions of transformed reads for each mirna from each group are shown in figure  <dig>  most mirnas had around 10- <dig> reads in these four groups.
there was no significant difference in the medians of the four libraries  indicating they had similar distributions of the mirna reads.
of the  <dig> distinct gallus gallus  mirna entries in mirbase  <cit> ,  <dig> mirnas were identified in chicken lungs and  <dig> mirnas in chicken tracheae.
in addition, we identified  <dig> potential novel mirna sequences .
seven novel mirnas were selected and all of them were confirmed by real-time quantification pcr in chicken lungs and tracheae, respectively, which is shown in table  <dig>
note:  <dig> expression levels were measured in terms of threshold cycle value  and normalized to u <dig> using 2-ΔΔct.
mirnas expression profiling analysis
in the present study there were four different groups: two tissues  and two states per tissue .
two comparisons were made between infected vs. non-infected within tissue and another two comparisons between lung and trachea at either infected or non-infected state.
differentially expressed mirnas in each comparison were identified .
within tissues,  <dig> and  <dig> mirnas were differentially expressed between infected and non-infected groups in lungs and tracheae, respectively .
when between tissues were compared,  <dig> and  <dig> mirnas were differentially expressed at infected and non-infected states, respectively .
note:  <dig> specifically expressed in infected lungs.
<dig> specifically expressed in non-infected lungs.
note:  <dig> specifically expressed in non-infected tracheae.
note:  <dig> specifically expressed in infected lungs.
note:  <dig> specifically expressed in non-infected lungs.
more mirnas  were down-regulated than up-regulated with aiv infection in both lungs and tracheae.
when infected vs. non-infected was compared,  <dig> mirnas  were only expressed in the non-infected lungs, while  <dig> mirnas  were only expressed in infected lungs .
between infected and non-infected tracheae, two  out of  <dig> down-regulated mirnas were expressed only in non-infected tracheae .
in the comparisons between tissues, only few mirnas  were highly expressed in tracheae compared to lungs .
under the infected state,  <dig> mirnas were specifically expressed in lungs and  <dig> mirnas were expressed at higher levels in lungs than in tracheae.
in the non-infected state,  <dig> mirnas specifically expressed in lungs and  <dig> mirnas were expressed at higher levels in lungs than tracheae.
of particular interest, mir-1a, mir- <dig>  and mir- <dig>  which were highly expressed in infected tracheas than the non-infected ones, and also were differentially expressed between infected tissues .
in the tissue comparison under the non-infected state, mir- <dig> was the only mirna that had higher expression level in tracheae than in lungs.
in general, those highly abundant mirnas were observed across all four groups examined .
note:  <dig> total number of reads for each group
confirmation of differentially expressed mirnas
taqman mirna assays were used to confirm the expression pattern of differentially expressed mirnas in lungs.
there were general consistency between taqman assay and deep sequence analysis in three mirnas  in terms of directions of regulation and significance.
specifically, there was a  <dig>  fold up-regulation  in mir-1a,  <dig>  fold down-regulation  in mir-125b, and  <dig>  fold down-regulation  in mir-146a .
clustering of chicken mirnas
chromosomal positions of differentially expressed mirnas revealed that some of them were very close to each other.
according to a previous report  <cit> , mirnas can be grouped as one cluster if they are less than  <dig>  bp apart on the same chromosome.
based on the mirbase  <dig>   <cit> , there are  <dig> mirna clusters in the chicken genome according to the criteria above.
eighteen of these clusters were detected in lungs and  <dig> clusters in tracheae, respectively .
each cluster contained at least two mirnas, and total of  <dig> mirnas were included in these clusters.
within these clusters, the mir-92-mir-19b-mir-20a-mir-19a-mir-18a-mir- <dig>  which is equivalent to the mammalian mir-17- <dig> cluster, and mir-302b-mir-302c-mir-1811-mir-302a-mir-302d-mir- <dig> cluster were the biggest clusters containing six mirnas.
both of them were detected in lungs.
there were only seven clusters differentially expressed .
clusters mir-16-1-mir-15a, let-7f-let-7a- <dig>  mir-181a-1-mir-181b- <dig>  let-7j-let-7k, mir-23b-mir-27b-mir- <dig>  and mir-16-2-mir-15b were down-regulated in lungs and mir-181a-1-mir-181b- <dig> was also down-regulated in tracheae with aiv infection.
cluster mir-34b-mir-34c was up-regulated in tracheae.
note:  <dig> identified in the library
<dig> not identified in the library
gene ontology analysis
potential target genes of differentially expressed mirnas in each comparison were predicted by miranda  <cit> .
in brief, each differentially expressed mirna was submitted to miranda individually and all of its targets predicted in miranda were used for the following gene ontology  analysis.
for each comparison, target genes of induced and repressed mirnas were separately analyzed.
all targets of induced mirnas in each comparison were submitted to david program  <cit>  and so were the targets of repressed mirnas.
functional category enrichment based on the go terms was evaluated on the targets of these differentially expressed mirnas.
all enriched go terms in biological process of each comparison were shown in additional file  <dig>  immune related go terms of each comparison are presented in figures  <dig>   <dig>   <dig> and  <dig>
in the comparison of infected vs. non-infected lungs,  <dig> immune related go terms in biological process were significantly enriched   from the targets of down-regulated mirnas.
response to virus was the go term with the highest fold enrichment .
other functional terms including immune system response, lymphocytes, and lung development were also identified.
in the comparison of infected vs. non-infected tracheae, six immune-related go terms in biological process were significantly enriched   from the targets of down-regulated mirnas, in which interleukin- <dig> production and interleukin- <dig> biosynthetic process had the highest fold enrichment .
for the targets of up-regulated mirnas between the infected vs. non-infected both in lung and trachea comparisons, only two immune related go terms: immunoglobulin i set and immunoglobulin subtype  <dig> in interpro category were significantly enriched.
for the tissue comparison under the infected state, targets of induced mirnas in lungs were associated with ten immune related go terms in biological process including tissue development, interleukin- <dig>  lymphocytes cytokines and immune system development  .
targets of repressed mirnas in lungs were associated with immune system development and immunoglobulin subtype  <dig> in the interpro category.
under the non-infected state,  <dig> immune related terms were significantly enriched in biological process in the targets of mirnas highly expressed in lungs.
these go terms were related to lung development and host immune system .
the go term nf-kappab binding was also enriched in molecular function .
discussion
the impact of mirnas expression on the understanding of molecular mechanisms in gene regulations has been remarkable.
although thousands of small rnas have been identified over the last decade, the challenge remains to fully identify all small nuclear rnas, especially very low abundant ones and to determine their individual functions.
the majority of known mirnas have been identified through traditional cloning method, which is both time consuming and labor intensive.
the advantages of next-generation sequencing technologies have provided an innovative tool to look into the genome with unprecedented depth of coverage.
solexa deep sequencing is one of these high throughput technologies, by which mirnas can be detected in any organism without prior sequence or secondary structure information.
this technology has been used in many species including human, mice and birds  <cit> .
expression of mirnas varies in different developmental stages  <cit> .
chicken mirnas identified in the present study provided novel information in the profiling of mirnas not only in aiv infected chickens, but also the two tissues  that have not been previously examined for mirna profiling in chickens.
to our knowledge, this is the first study to profile chicken mirnas in aiv infected chickens by deep sequencing approach.
there are  <dig> chicken mirnas predicted in mirbase  <dig>   <cit> .
the deep sequencing results in the current study experimentally confirmed  <dig> mirnas in chicken lungs and  <dig> mirnas in chicken tracheae, and the approach is more powerful than other conventional technologies previously used in birds  <cit> .
the identification of these chicken mirnas will be very useful in further investigating the functions and regulatory mechanisms of mirnas in the chicken.
growing evidence has suggested a relationship between differential mirna expression and human diseases  <cit> .
mirnas can regulate many aspects of the immune response, including the development and differentiation of b and t cells, proliferation of monocytes and neutrophils, antibody switching and the release of inflammatory mediators by regulating basic component of host immune system  <cit> .
mir- <dig> has been reported by several groups to play important roles in both innate and adaptive immune responses in mammals  <cit> .
mir- <dig> deficient mice lacked the capability to generate defensive immune responses and to develop lymphocytes, especially b-cell, and antigen-presenting cell functions  <cit> .
however, mir- <dig> showed very low abundance in both lungs and tracheae and no significant differential expression was observed in the present study.
over expression of mir-181a in mature mouse t cells can augment the sensitivity to peptide antigens, while suppressing mir-181a expression can reduce sensitivity and impair both positive and negative selection  <cit> .
selective expressions of mir-181a in the thymus and mir- <dig> in the bone marrow have been shown to be involved in the differentiation of pluripotent hematopoietic stem cells into the various blood cells lineages including b and t cells  <cit> .
in the present study, mir- <dig> was not significantly regulated while mir-181a was down-regulated in both infected lungs and tracheae.
in addition, mir-181a had a higher expression level in lungs than in tracheae under both infected and non-infected states.
the expression levels of mir-181a, 181a* and 181b were investigated in lps activated and cd40-lignad activated macrophages of chickens, respectively  <cit> .
only mir-181b was expressed in the macrophage cell line hd <dig> as well in the spleen adherent cells and that its expression increased after activation by lps or cd40-ligand  <cit> .
in the current study, mir-181b had same expression pattern with mir-181a in both lung and trachea comparisons.
these results suggest that mir-181a and mir-181b may be strong mirna candidates that regulate host response to aiv infection, and warrant further investigation of their targets and regulation mechanism in chickens.
although the interaction between mirna expression and virus infection remains to be elucidated, we speculated that mirna might target immune related genes or modulate virus replication.
sequencing of chicken mirnas in marek's disease virus  infected and non-infected chicken embryo fibroblast  indicated that more mirnas were up-regulated in mdv infected cells  <cit> .
these results differ from the current study in which most differentially expressed mirnas  were down-regulated in aiv infected tissues.
these results indicate that the mechanisms of mirna regulation of the host response to different types of virus in chickens are different.
chicken mir- <dig> and mir- <dig>  the most abundant mirnas in the cef small rna libraries, had significantly higher reads in mdv infected than non-infected cef  <cit> .
while both mir- <dig> and  <dig> had relatively lower abundance in the present study.
these results demonstrate that mirna expression can be tissue-specific with high abundance of mir- <dig> and  <dig> in the cef libraries and low abundances in lungs and tracheae.
it can also be speculated that host mirnas expression may be suppressed by aiv replication based on the mirna expression patterns observed in the current study.
some mirnas have been shown to be directly involved in virus replication.
a liver specific mirna  was shown to be required for hepatitis c virus  replication in humans  <cit> .
mir- <dig> can positively affect the viral replication and has become a therapeutic target for the treatment of hcv infection  <cit> .
in the current study, mir- <dig> specifically expressed in chicken lungs compared to tracheae under both infected and non-infected states.
these data suggest mir- <dig> might play a more important role in tissue distribution than the responses to aiv infection in chickens.
another two human mirnas  have potential target binding sites in polymerase basic  <dig>  and hemagglutinin  genes of aiv, respectively  <cit> .
unfortunately, these two mirnas are absent in the chicken genome, which might indicate different infectivity and lethality of the virus between chickens and humans.
although in the present study most differentially expressed mirna were down-regulated during aiv infection, some mirnas were also up-regulated.
mir-1a, mir- <dig> and mir- <dig> were significantly up-regulated in both tissues, while mir- <dig>  mir-34b and mir-34c were only up-regulated with aiv infection in tracheae.
this suggests different mirna regulation mechanisms might exist on host response to virus infection.
these up-regulated mirnas might inhibit gene expression of their target genes; therefore down-regulation of these target genes might help the host to inhibit virus replication.
different tissues serve different biological functions in animals and the expression patterns of mirnas can vary in different tissues  <cit> .
mirnas in bursa and spleen of developing chicken embryo have been recently identified, and diverse expression patterns of these mirnas between different immune organs were observed, suggesting that mirnas may function as dynamic regulators of the vertebrate immune system  <cit> .
some mirnas show tissue-specific distribution in mouse, suggesting specific functions within these tissues  <cit> .
in the current study, chicken lung and trachea were examined, as they are both part of the respiratory system and important sites for aiv replication.
there was a significant difference in mirna expression between lung and trachea with more mirnas expressed in lungs  than tracheae , although only small percentage of mirnas  were significantly differentially expressed in aiv infected samples.
when tissues in the state of virus infection were compared,  <dig> and  <dig> mirnas were specifically and highly expressed in lungs, respectively, and only  <dig> mirnas  were highly expressed in tracheae .
when tissues were compared under the non-infected state, all differentially expressed mirnas were expressed at higher levels in lungs than tracheae with the only exception of mir- <dig>  which showed a higher expression level in non-infected trachea than lung .
more interestingly, mir- <dig> was up-regulated in virus infected vs. non-infected lungs and was down-regulated in infected vs. non-infected tracheae.
we can conclude that mir- <dig> has an opposite regulatory role in lungs and tracheae or might have different targets in different tissues and therefore play different roles in host-virus interactions.
mir- <dig> and mir- <dig> were up-regulated in aiv infected chicken lungs, while they were specifically expressed in non-infected tracheae not the infected one .
the different regulation of mir- <dig> and mir- <dig> between lung and trachea suggests they may also have different mechanisms in response to aiv infection between tissues.
we hypothesize that mir-34b, mir-34c, mir- <dig>  mir- <dig> and mir- <dig> might be some of the most important mirnas associated with aiv infection.
significantly different mirna expression pattern between lung and trachea suggests the regulatory mechanism of mirnas on host response to the aiv infection between lung and trachea is distinct.
however, similar regulatory mechanism might also exist in these two tissues.
within the down-regulated mirnas in infected vs. non-infected lungs and tracheae, there were  <dig> mirnas which overlapped in both tissues.
this suggests that these  <dig> mirnas might have common modulation mechanisms with the aiv infection in chickens.
go term enrichment analysis has been widely used in functional analysis and allows the identification of important categories associated with functions of interests.
go terms enriched by the target genes of differentially expressed mirnas in the current study can provide useful information for the follow-up study to elucidate the regulatory mechanism of mirnas in host immune response to aiv infection.
during aiv infection, the host immune system is stimulated to develop a defensive mechanism, which might be the reason why genes involved in immune system development were enriched in all comparisons.
with virus infection, more immune related go terms were enriched by the targets of repressed mirnas in lungs than in tracheae  .
response to virus was identified as the most enriched term  in lung comparison, confirming that genes related to virus infection were regulated by mirnas.
the hyperinduction of proinflammatory cytokines such as tnf-α and ifn-β in human macrophages and respiratory epithelial cells by the highly pathogenic aiv h5n <dig> was believed to contribute to its high pathogenecity  <cit> .
lymphocytes were also reported to be suppressed by aiv  <cit> .
enrichments of t-cell and leukocytes activation and cytokines activities terms identified in the comparison of infected vs. non-infected lungs might be an indication of host immune system response against virus infection.
meanwhile, go terms involved in lung development and epithelium morphogenesis were enriched, suggesting the genes associated with lung epithelium development in lungs may be important for the recovery from aiv infection in chickens.
it was interesting that two go terms, interleuklin- <dig> production and biosynthetic process, were enriched in the infected tissue comparison, which were not included in the non-infected comparison.
these two terms were also enriched in the comparison of infected vs. non-infected trachea instead of the lung comparison.
il- <dig> plays a pivotal regulatory role in the anti-viral response due to its induction of ifn-γ, an anti-viral cytokine  <cit> .
these may suggest that a different defensive mechanism against virus infection might occur in trachea compared to lungs.
the two terms, response to virus and t-cell activation were also enriched by immune related genes differentially expressed in the early immune responses to h9n <dig> infection in tracheal organ cultures   <cit> .
host immune response, showed as adaptive immune responses in the current study, was enriched by differentially expressed genes in h5n <dig> infected chicken embryo fibroblasts  as well  <cit> .
influenza virus triggered a cascade of both innate and specific immune responses.
then both immune related genes and mirnas who might regulate these genes maybe involved in similar biological processes with the same go terms.
of special note, nf-kappab binding was also enriched in the comparison between lung and trachea under non-infected state.
a similar go term, regulation of nf-kappab, was enriched in the previous toc model with the infection of aiv h9n <dig>  <cit> .
activation of nf-kappab pathway is an essential immediate early step of immune activation.
many viruses have developed strategies to manipulate nf-kappab signalling through the use of multifunctional viral proteins that target the host innate immune response pathways  <cit> .
enrichment of go term nf-kappab binding suggests these two tissues might utilize this signal pathway differently.
post-transcriptional gene activity can be regulated through the interaction of regulatory rna-binding proteins and small non-coding rnas such as mirnas  <cit> .
mirnas can modulate protein activities by altering mrna stability, translational efficiency or localization  <cit> .
the 3' untranslated regions  are widely accepted as important post-transcriptional regulatory regions of mrnas, which are particularly rich in cis-acting regulatory elements  <cit> .
mirnas can regulate their target genes through the cis-acting regulatory elements  <cit> .
mirnas within the same cluster might share the same cis-regulatory elements  <cit> , and therefore, might have the same regulatory mechanism for their target genes.
out of the  <dig> mirna clusters identified in lungs and  <dig> mirna clusters identified in tracheae, there were  <dig> mirna clusters differentially expressed in different comparisons.
the mirnas from five of these clusters  identified in both lungs and tracheae were significantly down-regulated in infected lungs compared to non-infected lungs and also had higher expression levels in non-infected lungs than non-infected tracheae.
the mir-181a-1-mir181b- <dig> cluster was significantly down-regulated in both infected lungs and tracheae.
and the mir-34b-mir-34c cluster was the only significantly up-regulated cluster in the aiv infected trachea.
different mirna clusters had different regulation direction in aiv infected tissues in the present study.
this illustrates that, during aiv infection, different modulation mechanisms among different mirna clusters might coexist in both lungs and tracheae.
it is interesting to note that when considering the mirna clusters that were most active in chicken lung and trachea, mir-17- <dig> cluster  and mir-302b-mir-302c-mir-1811-mir-302a-mir-302d-mir- <dig> cluster are highly associated with cell proliferation and self-renewal of stem cells and cancer cells  <cit> .
in addition the mirnas clusters that were significantly down-regulated mir-15/ <dig> and let- <dig> are typically down-regulated in stem cells and cancer  <cit> .
these results suggest that aiv infection in chickens may instigate cell proliferation and self-renewal like behaviour in chicken lung epithelium and the newly recruited t lymphocytes.
modulation of target genes by mirna is one of most critical steps for gene expression regulation.
the targeted genes for some differentially expressed mirnas in the current study were predicted using miranda  <cit> .
interestingly, many of the target genes were involved in the host immune system.
the potential target genes for mir-1a and mir-1b are the t-cell immuno-modulatory protein.
mir-34b and mir34c, whose target genes are b-cell cll-pymphoma  <dig> &  <dig>  might be involved in the b-cell differentiation.
target genes for mir- <dig> were associated with monocyte macrophage differentiation, suggesting they maybe associated with antigen presentation.
based on other immune related mirna studies in mammals  <cit> , differentially expressed mirnas of their mammalian homologs and their targets are presented in table  <dig>  mir-15a, mir- <dig> and mir-181a have important functions in lymphocytes development and modulations while mir- <dig> and mir- <dig> are related to virus infection and mir-146a, induced by macrophages, can activate toll like receptor  and expose antigens to interleukin- <dig> beta.
although the exact functions of these mirnas in the aiv infected chickens remains to be determined, candidate mirnas and their potential targets identified in the current study provide strong evidence of their roles and warrant further investigation.
whether these chicken mirnas have the same function as mammals or not need to be validated in the future studies.
on-going efforts in the author's laboratory focusing on gene expressions of these target genes and determination of target genes for these differentially expressed mirnas will provide new insights of mirna regulations on aiv infection in chickens.
CONCLUSIONS
mirnas have recently been implicated in the intricate cross-talk between host and pathogen in viral infections and are critical in viral pathogenesis.
in aiv infected tissues, expression patterns of some host mirnas were significantly differentially regulated in the current study, supporting the hypothesis that certain mirnas are essential in the host-pathogen interactions.
once the role of these mirnas in the regulation of host-aiv interaction has been determined, it will improve the protective strategies in aiv infection in poultry.

 avian influenza virus is a type a virus of the family orthomyxovirida. although wild aquatic birds such as water fowls and sea gulls are their natural reservoir, land-based birds including chickens may also be infected, which cause significant economic losses to the poultry industry, and raise a great public health threat due to potential host jump from animals to humans. mirnas are non-coding, single-stranded rnas of 19~ nucleotides which represent a novel class of gene regulators and play important roles in a variety of biological processes in both plants and animals. mirnas modulate gene expression largely at the post-transcriptional level by different mechanisms including direct cleavage of targeted mrnas, inhibition of translation or even up-regulation of translation. mirnas are involved in different biological activities such as development, differentiation, growth and metabolism. recently, in mammals, mirnas have been reported to participate in the regulation of immunity, including development and differentiation of lymphocytes, monocytes and neutrophils, and modulation of inflammation. mir- expresses in mature b and t cells derived from mouse hematopoietic stem cells, and is able to block early b cell development when expressed prematurely. mir-181a is an intrinsic modulator of t cell sensitivity and selection in mice. after exposure of thp- cells to lipopolysaccharides, mir- was identified as an inhibitor of signalling proteins of the innate immune responses by nf-kappab. mirnas have also been found to be critical effectors in the regulation of viral pathogenesis. two human encoded mirnas, have been shown to have potential binding sites for the genes that code for the polymerase basic and hemmagglutinin proteins and are reported to be involved in the pathogenesis of h5n aiv. all of these evidences suggest that certain mirnas might be important in the modulation of aiv infections in chickens. in order to effectively control aiv infection in poultry, it is essential to elucidate the mechanisms of virus pathogenesis in chickens. however, how host cells interact with aivs during infection in poultry remains poorly understood. identification of differentially expressed mirnas in aiv infected chickens will pave a novel avenue to understand host-virus interaction. with the development of next generation sequencing, massively parallel sequencing holds great promise for expression profiling and it can provide a superior sensitivity at high sequencing depth to discover especially those mirnas with low abundance and novel mirnas that are not able to be identified using traditional cloning approaches. deep sequencing has been previously used to profile both chicken mirnas and marek's disease virus mirnas. in the current study, a solexa sequencer was used to deep sequence differentially regulated chicken mirnas in h5n infected and non-infected spf chickens. our results will expand the list of mirnas which might be related to the host immune responses in animals.  virus titration virus replication was examined by real-time rt-pcr for influenza matrix gene from total rnas of lung and tracheae at dpi. the titer of infected samples was log eid50/ml in lung, and log eid50/ml in tracheae. both non-infected lung and tracheae samples were negative. small rna libraries a total of and filtered high quality reads were obtained from chicken lungs and tracheae, respectively. in the libraries of chicken lungs, and reads were obtained from infected and non-infected lungs, respectively. out of these reads, of these high quality reads were exact matches while another reads were loose matches to known chicken mirnas. all reads with a perfect match to mature mirna sequences from chicken deposited in mirbase with insertions or deletions of 1- nucleotides was considered as a loose match to represent dicer-processing products from each of the chicken mirna precursors. an example from gga-mir-181a is shown in figure here we saw that the sample had copies of a sequence that was identical to that of the mature gga-mir-181a. in addition we observed significant alternate processing at the 3'-end that was characteristic of mirnas in various copy numbers. loose matches were defined by sequence reads that aligned with chicken mirna consensus sequence with 1- mismatches. these may represent sequencing errors, mutations and/or rna editing events. in the libraries of chicken trachea, reads were obtained from infected tracheae and were obtained from non-infected tracheae. out of these reads, of these high quality reads were exact matches and another reads were loose matches to known chicken mirnas. the sum of exact and loose match reads was used as the total number of reads for each mirna. to display the distribution of mirnas at each library, reads of individual mirna within each group were transformed to log the plots of distributions of transformed reads for each mirna from each group are shown in figure most mirnas had around 10- reads in these four groups. there was no significant difference in the medians of the four libraries indicating they had similar distributions of the mirna reads. of the distinct gallus gallus mirna entries in mirbase, mirnas were identified in chicken lungs and mirnas in chicken tracheae. in addition, we identified potential novel mirna sequences. seven novel mirnas were selected and all of them were confirmed by real-time quantification pcr in chicken lungs and tracheae, respectively, which is shown in table note: expression levels were measured in terms of threshold cycle value and normalized to u using 2-ΔΔct. mirnas expression profiling analysis in the present study there were four different groups: two tissues and two states per tissue. two comparisons were made between infected vs. non-infected within tissue and another two comparisons between lung and trachea at either infected or non-infected state. differentially expressed mirnas in each comparison were identified. within tissues, and mirnas were differentially expressed between infected and non-infected groups in lungs and tracheae, respectively. when between tissues were compared, and mirnas were differentially expressed at infected and non-infected states, respectively. note: specifically expressed in infected lungs. specifically expressed in non-infected lungs. note: specifically expressed in non-infected tracheae. note: specifically expressed in infected lungs. note: specifically expressed in non-infected lungs. more mirnas were down-regulated than up-regulated with aiv infection in both lungs and tracheae. when infected vs. non-infected was compared, mirnas were only expressed in the non-infected lungs, while mirnas were only expressed in infected lungs. between infected and non-infected tracheae, two out of down-regulated mirnas were expressed only in non-infected tracheae. in the comparisons between tissues, only few mirnas were highly expressed in tracheae compared to lungs. under the infected state, mirnas were specifically expressed in lungs and mirnas were expressed at higher levels in lungs than in tracheae. in the non-infected state, mirnas specifically expressed in lungs and mirnas were expressed at higher levels in lungs than tracheae. of particular interest, mir-1a, mir- and mir- which were highly expressed in infected tracheas than the non-infected ones, and also were differentially expressed between infected tissues. in the tissue comparison under the non-infected state, mir- was the only mirna that had higher expression level in tracheae than in lungs. in general, those highly abundant mirnas were observed across all four groups examined. note: total number of reads for each group confirmation of differentially expressed mirnas taqman mirna assays were used to confirm the expression pattern of differentially expressed mirnas in lungs. there were general consistency between taqman assay and deep sequence analysis in three mirnas in terms of directions of regulation and significance. specifically, there was a fold up-regulation in mir-1a, fold down-regulation in mir-125b, and fold down-regulation in mir-146a. clustering of chicken mirnas chromosomal positions of differentially expressed mirnas revealed that some of them were very close to each other. according to a previous report, mirnas can be grouped as one cluster if they are less than bp apart on the same chromosome. based on the mirbase, there are mirna clusters in the chicken genome according to the criteria above. eighteen of these clusters were detected in lungs and clusters in tracheae, respectively. each cluster contained at least two mirnas, and total of mirnas were included in these clusters. within these clusters, the mir-92-mir-19b-mir-20a-mir-19a-mir-18a-mir- which is equivalent to the mammalian mir-17- cluster, and mir-302b-mir-302c-mir-1811-mir-302a-mir-302d-mir- cluster were the biggest clusters containing six mirnas. both of them were detected in lungs. there were only seven clusters differentially expressed. clusters mir-16-1-mir-15a, let-7f-let-7a- mir-181a-1-mir-181b- let-7j-let-7k, mir-23b-mir-27b-mir- and mir-16-2-mir-15b were down-regulated in lungs and mir-181a-1-mir-181b- was also down-regulated in tracheae with aiv infection. cluster mir-34b-mir-34c was up-regulated in tracheae. note: identified in the library not identified in the library gene ontology analysis potential target genes of differentially expressed mirnas in each comparison were predicted by miranda. in brief, each differentially expressed mirna was submitted to miranda individually and all of its targets predicted in miranda were used for the following gene ontology analysis. for each comparison, target genes of induced and repressed mirnas were separately analyzed. all targets of induced mirnas in each comparison were submitted to david program and so were the targets of repressed mirnas. functional category enrichment based on the go terms was evaluated on the targets of these differentially expressed mirnas. all enriched go terms in biological process of each comparison were shown in additional file immune related go terms of each comparison are presented in figures and in the comparison of infected vs. non-infected lungs, immune related go terms in biological process were significantly enriched from the targets of down-regulated mirnas. response to virus was the go term with the highest fold enrichment. other functional terms including immune system response, lymphocytes, and lung development were also identified. in the comparison of infected vs. non-infected tracheae, six immune-related go terms in biological process were significantly enriched from the targets of down-regulated mirnas, in which interleukin- production and interleukin- biosynthetic process had the highest fold enrichment. for the targets of up-regulated mirnas between the infected vs. non-infected both in lung and trachea comparisons, only two immune related go terms: immunoglobulin i set and immunoglobulin subtype in interpro category were significantly enriched. for the tissue comparison under the infected state, targets of induced mirnas in lungs were associated with ten immune related go terms in biological process including tissue development, interleukin- lymphocytes cytokines and immune system development. targets of repressed mirnas in lungs were associated with immune system development and immunoglobulin subtype in the interpro category. under the non-infected state, immune related terms were significantly enriched in biological process in the targets of mirnas highly expressed in lungs. these go terms were related to lung development and host immune system. the go term nf-kappab binding was also enriched in molecular function. discussion the impact of mirnas expression on the understanding of molecular mechanisms in gene regulations has been remarkable. although thousands of small rnas have been identified over the last decade, the challenge remains to fully identify all small nuclear rnas, especially very low abundant ones and to determine their individual functions. the majority of known mirnas have been identified through traditional cloning method, which is both time consuming and labor intensive. the advantages of next-generation sequencing technologies have provided an innovative tool to look into the genome with unprecedented depth of coverage. solexa deep sequencing is one of these high throughput technologies, by which mirnas can be detected in any organism without prior sequence or secondary structure information. this technology has been used in many species including human, mice and birds. expression of mirnas varies in different developmental stages. chicken mirnas identified in the present study provided novel information in the profiling of mirnas not only in aiv infected chickens, but also the two tissues that have not been previously examined for mirna profiling in chickens. to our knowledge, this is the first study to profile chicken mirnas in aiv infected chickens by deep sequencing approach. there are chicken mirnas predicted in mirbase. the deep sequencing results in the current study experimentally confirmed mirnas in chicken lungs and mirnas in chicken tracheae, and the approach is more powerful than other conventional technologies previously used in birds. the identification of these chicken mirnas will be very useful in further investigating the functions and regulatory mechanisms of mirnas in the chicken. growing evidence has suggested a relationship between differential mirna expression and human diseases. mirnas can regulate many aspects of the immune response, including the development and differentiation of b and t cells, proliferation of monocytes and neutrophils, antibody switching and the release of inflammatory mediators by regulating basic component of host immune system. mir- has been reported by several groups to play important roles in both innate and adaptive immune responses in mammals. mir- deficient mice lacked the capability to generate defensive immune responses and to develop lymphocytes, especially b-cell, and antigen-presenting cell functions. however, mir- showed very low abundance in both lungs and tracheae and no significant differential expression was observed in the present study. over expression of mir-181a in mature mouse t cells can augment the sensitivity to peptide antigens, while suppressing mir-181a expression can reduce sensitivity and impair both positive and negative selection. selective expressions of mir-181a in the thymus and mir- in the bone marrow have been shown to be involved in the differentiation of pluripotent hematopoietic stem cells into the various blood cells lineages including b and t cells. in the present study, mir- was not significantly regulated while mir-181a was down-regulated in both infected lungs and tracheae. in addition, mir-181a had a higher expression level in lungs than in tracheae under both infected and non-infected states. the expression levels of mir-181a, 181a* and 181b were investigated in lps activated and cd40-lignad activated macrophages of chickens, respectively. only mir-181b was expressed in the macrophage cell line hd as well in the spleen adherent cells and that its expression increased after activation by lps or cd40-ligand. in the current study, mir-181b had same expression pattern with mir-181a in both lung and trachea comparisons. these results suggest that mir-181a and mir-181b may be strong mirna candidates that regulate host response to aiv infection, and warrant further investigation of their targets and regulation mechanism in chickens. although the interaction between mirna expression and virus infection remains to be elucidated, we speculated that mirna might target immune related genes or modulate virus replication. sequencing of chicken mirnas in marek's disease virus infected and non-infected chicken embryo fibroblast indicated that more mirnas were up-regulated in mdv infected cells. these results differ from the current study in which most differentially expressed mirnas were down-regulated in aiv infected tissues. these results indicate that the mechanisms of mirna regulation of the host response to different types of virus in chickens are different. chicken mir- and mir- the most abundant mirnas in the cef small rna libraries, had significantly higher reads in mdv infected than non-infected cef. while both mir- and had relatively lower abundance in the present study. these results demonstrate that mirna expression can be tissue-specific with high abundance of mir- and in the cef libraries and low abundances in lungs and tracheae. it can also be speculated that host mirnas expression may be suppressed by aiv replication based on the mirna expression patterns observed in the current study. some mirnas have been shown to be directly involved in virus replication. a liver specific mirna was shown to be required for hepatitis c virus replication in humans. mir- can positively affect the viral replication and has become a therapeutic target for the treatment of hcv infection. in the current study, mir- specifically expressed in chicken lungs compared to tracheae under both infected and non-infected states. these data suggest mir- might play a more important role in tissue distribution than the responses to aiv infection in chickens. another two human mirnas have potential target binding sites in polymerase basic and hemagglutinin genes of aiv, respectively. unfortunately, these two mirnas are absent in the chicken genome, which might indicate different infectivity and lethality of the virus between chickens and humans. although in the present study most differentially expressed mirna were down-regulated during aiv infection, some mirnas were also up-regulated. mir-1a, mir- and mir- were significantly up-regulated in both tissues, while mir- mir-34b and mir-34c were only up-regulated with aiv infection in tracheae. this suggests different mirna regulation mechanisms might exist on host response to virus infection. these up-regulated mirnas might inhibit gene expression of their target genes; therefore down-regulation of these target genes might help the host to inhibit virus replication. different tissues serve different biological functions in animals and the expression patterns of mirnas can vary in different tissues. mirnas in bursa and spleen of developing chicken embryo have been recently identified, and diverse expression patterns of these mirnas between different immune organs were observed, suggesting that mirnas may function as dynamic regulators of the vertebrate immune system. some mirnas show tissue-specific distribution in mouse, suggesting specific functions within these tissues. in the current study, chicken lung and trachea were examined, as they are both part of the respiratory system and important sites for aiv replication. there was a significant difference in mirna expression between lung and trachea with more mirnas expressed in lungs than tracheae, although only small percentage of mirnas were significantly differentially expressed in aiv infected samples. when tissues in the state of virus infection were compared, and mirnas were specifically and highly expressed in lungs, respectively, and only mirnas were highly expressed in tracheae. when tissues were compared under the non-infected state, all differentially expressed mirnas were expressed at higher levels in lungs than tracheae with the only exception of mir- which showed a higher expression level in non-infected trachea than lung. more interestingly, mir- was up-regulated in virus infected vs. non-infected lungs and was down-regulated in infected vs. non-infected tracheae. we can conclude that mir- has an opposite regulatory role in lungs and tracheae or might have different targets in different tissues and therefore play different roles in host-virus interactions. mir- and mir- were up-regulated in aiv infected chicken lungs, while they were specifically expressed in non-infected tracheae not the infected one. the different regulation of mir- and mir- between lung and trachea suggests they may also have different mechanisms in response to aiv infection between tissues. we hypothesize that mir-34b, mir-34c, mir- mir- and mir- might be some of the most important mirnas associated with aiv infection. significantly different mirna expression pattern between lung and trachea suggests the regulatory mechanism of mirnas on host response to the aiv infection between lung and trachea is distinct. however, similar regulatory mechanism might also exist in these two tissues. within the down-regulated mirnas in infected vs. non-infected lungs and tracheae, there were mirnas which overlapped in both tissues. this suggests that these mirnas might have common modulation mechanisms with the aiv infection in chickens. go term enrichment analysis has been widely used in functional analysis and allows the identification of important categories associated with functions of interests. go terms enriched by the target genes of differentially expressed mirnas in the current study can provide useful information for the follow-up study to elucidate the regulatory mechanism of mirnas in host immune response to aiv infection. during aiv infection, the host immune system is stimulated to develop a defensive mechanism, which might be the reason why genes involved in immune system development were enriched in all comparisons. with virus infection, more immune related go terms were enriched by the targets of repressed mirnas in lungs than in tracheae. response to virus was identified as the most enriched term in lung comparison, confirming that genes related to virus infection were regulated by mirnas. the hyperinduction of proinflammatory cytokines such as tnf-α and ifn-β in human macrophages and respiratory epithelial cells by the highly pathogenic aiv h5n was believed to contribute to its high pathogenecity. lymphocytes were also reported to be suppressed by aiv. enrichments of t-cell and leukocytes activation and cytokines activities terms identified in the comparison of infected vs. non-infected lungs might be an indication of host immune system response against virus infection. meanwhile, go terms involved in lung development and epithelium morphogenesis were enriched, suggesting the genes associated with lung epithelium development in lungs may be important for the recovery from aiv infection in chickens. it was interesting that two go terms, interleuklin- production and biosynthetic process, were enriched in the infected tissue comparison, which were not included in the non-infected comparison. these two terms were also enriched in the comparison of infected vs. non-infected trachea instead of the lung comparison. il- plays a pivotal regulatory role in the anti-viral response due to its induction of ifn-γ, an anti-viral cytokine. these may suggest that a different defensive mechanism against virus infection might occur in trachea compared to lungs. the two terms, response to virus and t-cell activation were also enriched by immune related genes differentially expressed in the early immune responses to h9n infection in tracheal organ cultures. host immune response, showed as adaptive immune responses in the current study, was enriched by differentially expressed genes in h5n infected chicken embryo fibroblasts as well. influenza virus triggered a cascade of both innate and specific immune responses. then both immune related genes and mirnas who might regulate these genes maybe involved in similar biological processes with the same go terms. of special note, nf-kappab binding was also enriched in the comparison between lung and trachea under non-infected state. a similar go term, regulation of nf-kappab, was enriched in the previous toc model with the infection of aiv h9n. activation of nf-kappab pathway is an essential immediate early step of immune activation. many viruses have developed strategies to manipulate nf-kappab signalling through the use of multifunctional viral proteins that target the host innate immune response pathways. enrichment of go term nf-kappab binding suggests these two tissues might utilize this signal pathway differently. post-transcriptional gene activity can be regulated through the interaction of regulatory rna-binding proteins and small non-coding rnas such as mirnas. mirnas can modulate protein activities by altering mrna stability, translational efficiency or localization. the 3' untranslated regions are widely accepted as important post-transcriptional regulatory regions of mrnas, which are particularly rich in cis-acting regulatory elements. mirnas can regulate their target genes through the cis-acting regulatory elements. mirnas within the same cluster might share the same cis-regulatory elements, and therefore, might have the same regulatory mechanism for their target genes. out of the mirna clusters identified in lungs and mirna clusters identified in tracheae, there were mirna clusters differentially expressed in different comparisons. the mirnas from five of these clusters identified in both lungs and tracheae were significantly down-regulated in infected lungs compared to non-infected lungs and also had higher expression levels in non-infected lungs than non-infected tracheae. the mir-181a-1-mir181b- cluster was significantly down-regulated in both infected lungs and tracheae. and the mir-34b-mir-34c cluster was the only significantly up-regulated cluster in the aiv infected trachea. different mirna clusters had different regulation direction in aiv infected tissues in the present study. this illustrates that, during aiv infection, different modulation mechanisms among different mirna clusters might coexist in both lungs and tracheae. it is interesting to note that when considering the mirna clusters that were most active in chicken lung and trachea, mir-17- cluster and mir-302b-mir-302c-mir-1811-mir-302a-mir-302d-mir- cluster are highly associated with cell proliferation and self-renewal of stem cells and cancer cells. in addition the mirnas clusters that were significantly down-regulated mir-15/ and let- are typically down-regulated in stem cells and cancer. these results suggest that aiv infection in chickens may instigate cell proliferation and self-renewal like behaviour in chicken lung epithelium and the newly recruited t lymphocytes. modulation of target genes by mirna is one of most critical steps for gene expression regulation. the targeted genes for some differentially expressed mirnas in the current study were predicted using miranda. interestingly, many of the target genes were involved in the host immune system. the potential target genes for mir-1a and mir-1b are the t-cell immuno-modulatory protein. mir-34b and mir34c, whose target genes are b-cell cll-pymphoma & might be involved in the b-cell differentiation. target genes for mir- were associated with monocyte macrophage differentiation, suggesting they maybe associated with antigen presentation. based on other immune related mirna studies in mammals, differentially expressed mirnas of their mammalian homologs and their targets are presented in table mir-15a, mir- and mir-181a have important functions in lymphocytes development and modulations while mir- and mir- are related to virus infection and mir-146a, induced by macrophages, can activate toll like receptor and expose antigens to interleukin- beta. although the exact functions of these mirnas in the aiv infected chickens remains to be determined, candidate mirnas and their potential targets identified in the current study provide strong evidence of their roles and warrant further investigation. whether these chicken mirnas have the same function as mammals or not need to be validated in the future studies. on-going efforts in the author's laboratory focusing on gene expressions of these target genes and determination of target genes for these differentially expressed mirnas will provide new insights of mirna regulations on aiv infection in chickens.  mirnas have recently been implicated in the intricate cross-talk between host and pathogen in viral infections and are critical in viral pathogenesis. in aiv infected tissues, expression patterns of some host mirnas were significantly differentially regulated in the current study, supporting the hypothesis that certain mirnas are essential in the host-pathogen interactions. once the role of these mirnas in the regulation of host-aiv interaction has been determined, it will improve the protective strategies in aiv infection in poultry. 
the objective of this study was to identify mirnas associated with aiv infections in chickens. elucidation of the mechanism of these mirnas on the regulation of host-aiv interaction will lead to the development of new control strategies to prevent or treat aiv infections in poultry. total rnas were isolated from lung and trachea of low pathogenic h5n infected and non-infected spf chickens at days post-infection. avian influenza virus not only causes significant economic losses in poultry production, but also is of great concern to human health. micrornas play critical roles in a wide spectrum of biological processes and have been shown to be important effectors in the intricate host-pathogen interaction networks. during aiv infection, many host mirnas were differentially regulated, supporting the hypothesis that certain mirnas might be essential in the host-pathogen interactions.
 micrornas play critical roles in a wide spectrum of biological processes and have been shown to be important effectors in the intricate host-pathogen interaction networks. avian influenza virus not only causes significant economic losses in poultry production, but also is of great concern to human health. the objective of this study was to identify mirnas associated with aiv infections in chickens.  total rnas were isolated from lung and trachea of low pathogenic h5n infected and non-infected spf chickens at days post-infection. a total of and reads were obtained from lung and trachea, respectively. and mirnas were detected in lungs and in tracheae from a total of distinct chicken mirnas available at the mirbase, respectively. seventy-three and thirty-six mirnas were differentially expressed between infected and non-infected chickens in lungs and tracheae, respectively. there were more mirnas highly expressed in non-infected tissues than in infected tissues. interestingly, some of these differentially expressed mirnas, including mir- have been previously reported to be associated with immune-related signal pathways in mammals.  to our knowledge, this is the first study on mirna gene expression in aiv infected chickens using a deep sequencing approach. during aiv infection, many host mirnas were differentially regulated, supporting the hypothesis that certain mirnas might be essential in the host-pathogen interactions. elucidation of the mechanism of these mirnas on the regulation of host-aiv interaction will lead to the development of new control strategies to prevent or treat aiv infections in poultry. 