hepatitis b virus ( hbv ) is a hepatotrophic virus that causes a major global health problem . an estimated 2 billion individuals have been infected with hbv and approximately 350 million have the chronic disease . although the mechanism of hbv pathogenesis remains elusive , host genetic factors are proposed to govern the pathology of disease progression or regression , along with viral and environmental factors . nk cells are activated in the early response to infection , and there is substantial population variability in the rates of hbv infection . killer cell immunoglobulin - like receptors ( kirs ) are members of the group of cell - surface molecules that activate or inhibit nk cell interaction with hla class i molecules on the surface of target cells . although detailed genetic and functional analyses exploring kir influences on hbv in large cohorts are lacking , accumulating evidence supports that nk cell activation contributes to inflammation and liver injury during hbv infection both in hbv transgenic mice and in hbv infected patients [ 58 ] . kirs are members of the immunoglobulin superfamily of receptors and are encoded on chromosome 19q13.4 . the kir gene cluster comprises up to 16 highly homologous and closely linked genes and pseudogenes . fourteen of them encode receptors triggering either inhibition ( 3dl13 , 2dl13 , 2dl5 ) or activation ( 3ds1 , 2ds15 ) and/or both ( 2dl4 ) , and 2 pseudogenes ( 2dp1 and 3dp1 ) do not encode cell - surface receptors . encode for cell - surface receptors , while the remaining alleles , 2ds4 * 003/4/6/7/8/9 , carry a 22-base pair deletion in exon 5 , which causes a frame shift , yielding a truncated kir2ds4 protein with loss of the transmembrane and cytoplasmic domains of the full - length kir2ds4 protein . additionally , different combinations of kir genes generate inherited haplotypes that can be divided into 2 basic groups  a and b  on the basis of their gene content . polymorphic kirs , which interact with hla class 1 , are largely inhibitory and exhibit substantial genetic diversity . the result is a significant variation of nk cell repertoire between individuals and also between populations . as each kir - ligand interaction may have differential effects on nk cell activation and inhibition , this diversity has important potential influences on the host response to infections . genetic studies have demonstrated associations between specific kir - ligand combinations and the pathogenesis and progression of diverse viral infectious diseases [ 1217 ] . there is still limited data on the relationship of kir genes and chronic hbv infection in the literature . additionally , to our knowledge , no analogous study has been performed in our population thus far . the aim of this study was to analyze whether inhibiting or activatory kir genes and different kir genotypes have an association in the progression of hbv infection in a turkish population . this may help to explore some of the possible immune genes that could be important in predisposition to chronic hbv infection . the patient groups consisted of 37 patients ( 17 male , 20 female ) with chronic hbv infection and 36 patients ( 15 male , 21 female ) in spontaneous remission state of hbv infection diagnosed at the department of infectious diseases of the cukurova university , balcal hospital . the diagnostic criterion for chronic hbv adopted in this study the control group consisted of 85 healthy subjects ( 38 males and 47 females ) . the ages ranged from 18 to 70 years ( mean , 43 years ) for the patients with chronic hbv , from 19 to 78 years ( mean , 42.6 years ) for the patients with spontaneous remission , and from 19 to 74 years ( mean , 42.5 years ) for the control group . all individuals included in this study were from the cukurova region of turkey and they were all matched for ethnicity . all the enrolled subjects had no serological evidence of hepatitis c virus , hepatitis d virus , and hiv infections , and had no other diseases such as diabetes , malignant tumor , or autoimmune diseases . samples were collected after informed written consent that was obtained from all participants , and the study was approved by the cukurova university ethics committee . diagnosis of chronic hbv infection was based on seropositivity for anti - hbv antibody using electrochemiluminescence immunoassay ( roche diagnostics , gmbh , mannheim , germany ) and the confirmation of hbv dna using light cycler 2.0 real - time polymerase chain reaction ( roche diagnostics , gmbh , mannheim , germany ) . dna from a venous blood sample of each subject was extracted by dna isolation kit ( qiaamp dna blood mini kit , cat no : 51104 , qiagen vertriebs gmbh , vienna , austria ) . genotyping of kir genes was performed using the multiplex kir - sso typing kit from tepnel lifecodes corporation ( ref : 545110r , ct , usa ) . this product consists of a mixture of locus - specific oligonucleotide probes coupled to color - coded microspheres ( luminex corp ) and 2 pcr reactions for the amplification of kir - exons 4 , 5 , 7 , 8 , and 9 . to type each sample , pcr was performed and the product was hybridized with the sso - probe mixture using the manufacturer s protocol . frequencies of group - a and -b kir haplotypes were deduced from the genotype data . in individuals carrying only kir3dl3 , 2dl3 , 2dl1 , 2dp1 , 3dp1 , 2dl4 , 3dl1 , 2ds4 and 3dl2 , a fixed - gene content characteristic of group - a haplotypes was considered carrying 2 copies of group - a kir haplotypes ( aa genotypes ) . if any of genes kir2dl2 , 2dl5 , 3ds1 , 2ds1 , 2ds2 , 2ds3 and/or 2ds5 were present , then the genotype was considered as having b haplotype ( bx ) . the percentage of each kir gene in the patient and control groups was determined by direct counting ( individuals positive for the gene / individuals tested per population100 ) . differences between 2 groups in the distribution of each kir gene were estimated by 2-tailed fisher s exact test and p<0.05 was considered to be statistically significant . the patient groups consisted of 37 patients ( 17 male , 20 female ) with chronic hbv infection and 36 patients ( 15 male , 21 female ) in spontaneous remission state of hbv infection diagnosed at the department of infectious diseases of the cukurova university , balcal hospital . the diagnostic criterion for chronic hbv adopted in this study the control group consisted of 85 healthy subjects ( 38 males and 47 females ) . the ages ranged from 18 to 70 years ( mean , 43 years ) for the patients with chronic hbv , from 19 to 78 years ( mean , 42.6 years ) for the patients with spontaneous remission , and from 19 to 74 years ( mean , 42.5 years ) for the control group . all individuals included in this study were from the cukurova region of turkey and they were all matched for ethnicity . all the enrolled subjects had no serological evidence of hepatitis c virus , hepatitis d virus , and hiv infections , and had no other diseases such as diabetes , malignant tumor , or autoimmune diseases . samples were collected after informed written consent that was obtained from all participants , and the study was approved by the cukurova university ethics committee . diagnosis of chronic hbv infection was based on seropositivity for anti - hbv antibody using electrochemiluminescence immunoassay ( roche diagnostics , gmbh , mannheim , germany ) and the confirmation of hbv dna using light cycler 2.0 real - time polymerase chain reaction ( roche diagnostics , gmbh , mannheim , germany ) . dna from a venous blood sample of each subject was extracted by dna isolation kit ( qiaamp dna blood mini kit , cat no : 51104 , qiagen vertriebs gmbh , vienna , austria ) . genotyping of kir genes was performed using the multiplex kir - sso typing kit from tepnel lifecodes corporation ( ref : 545110r , ct , usa ) . this product consists of a mixture of locus - specific oligonucleotide probes coupled to color - coded microspheres ( luminex corp ) and 2 pcr reactions for the amplification of kir - exons 4 , 5 , 7 , 8 , and 9 . to type each sample , pcr was performed and the product was hybridized with the sso - probe mixture using the manufacturer s protocol . frequencies of group - a and -b kir haplotypes were deduced from the genotype data . in individuals carrying only kir3dl3 , 2dl3 , 2dl1 , 2dp1 , 3dp1 , 2dl4 , 3dl1 , 2ds4 and 3dl2 , a fixed - gene content characteristic of group - a haplotypes was considered carrying 2 copies of group - a kir haplotypes ( aa genotypes ) . if any of genes kir2dl2 , 2dl5 , 3ds1 , 2ds1 , 2ds2 , 2ds3 and/or 2ds5 were present , then the genotype was considered as having b haplotype ( bx ) . the percentage of each kir gene in the patient and control groups was determined by direct counting ( individuals positive for the gene / individuals tested per population100 ) . differences between 2 groups in the distribution of each kir gene were estimated by 2-tailed fisher s exact test and p<0.05 was considered to be statistically significant . framework genes kir2dl4 , 3dl2 , 3dl3 , and 3dp1 were present in all of the samples ( figure 1 ) . among inhibitory kir genes , 2dl1 and 3dl1 had higher frequencies in all samples of chronic hbv patients , which were more than 78.4% . with the exception of kir2ds4 , frequencies of the remaining activating genes were all lower than 54.1% ( table 1 ) . the most frequent genotype , found in 13.5% of patients , was aa180 genotype , which consists of only 1 activating gene other patients demonstrated the presence of more than 1 activating gene and thus were considered as bx ( 67.6% ) . two different aa genotypes  aa180 ( 13.9% ) and aa1 ( 8.3% )  were found in the spontaneous remission group , which consists of only one activating gene  kir2ds4 ( figure 2 ) . other individuals in this group demonstrated the presence of more than 1 activating gene and thus were considered as bx ( 76.8% ) . framework genes kir2dl4 , 3dl2 , 3dl3 , and 3dp1 were also present in all of the samples ( figure 2 ) . among inhibitory kir genes , 2dl1 and 3dl1 had higher frequencies in all samples of chronic hbv patients , which were more than 80.6% . with the exception of kir2ds4 , frequencies of the remaining activating genes were all lower than 63.9% ( table 1 ) . in the first step , frequencies of individual kir genes were compared between chronic hbv patients and patients with spontaneous remission ( table 1 ) . in the second step , frequencies of individual kir genes were compared between the control group and the group composed of chronic hbv patients and patients with spontaneous remission ( table 2 , figure 3 ) . the rate of inhibitory kir2dl3 ( p=0.0 ) and 3ds1 ( p=0.0 ) were higher in the healthy group than the group composed of chronic hbv patients and patients with spontaneous remission . since the methods used for kir genotyping permitted distinguishing groups of alleles of activating kir2ds4 , we found that there were no statistically significant differences for 2ds4 * 001 ( full - length exon 5 ) and 2ds4 * 003/4/6/7/8/9 ( deletion in exon 5 ) alleles between the 2 groups ( p>0.05 ) . additionally , there were no statistically significant differences between the rate of aa and bx genotypes of chronic hbv patients and patients with spontaneous remission and the control group ( p>0.05 ) . framework genes kir2dl4 , 3dl2 , 3dl3 , and 3dp1 were present in all of the samples ( figure 1 ) . among inhibitory kir genes , 2dl1 and 3dl1 had higher frequencies in all samples of chronic hbv patients , which were more than 78.4% . with the exception of kir2ds4 , frequencies of the remaining activating genes were all lower than 54.1% ( table 1 ) . the most frequent genotype , found in 13.5% of patients , was aa180 genotype , which consists of only 1 activating gene other patients demonstrated the presence of more than 1 activating gene and thus were considered as bx ( 67.6% ) . two different aa genotypes  aa180 ( 13.9% ) and aa1 ( 8.3% )  were found in the spontaneous remission group , which consists of only one activating gene  kir2ds4 ( figure 2 ) . other individuals in this group demonstrated the presence of more than 1 activating gene and thus were considered as bx ( 76.8% ) . framework genes kir2dl4 , 3dl2 , 3dl3 , and 3dp1 were also present in all of the samples ( figure 2 ) . among inhibitory kir genes , 2dl1 and 3dl1 had higher frequencies in all samples of chronic hbv patients , which were more than 80.6% . with the exception of kir2ds4 , frequencies of the remaining activating genes were all lower than 63.9% ( table 1 ) . in the first step , frequencies of individual kir genes were compared between chronic hbv patients and patients with spontaneous remission ( table 1 ) . there were no statistically significant differences between these 2 groups . in the second step , frequencies of individual kir genes were compared between the control group and the group composed of chronic hbv patients and patients with spontaneous remission ( table 2 , figure 3 ) . the rate of inhibitory kir2dl3 ( p=0.0 ) and 3ds1 ( p=0.0 ) were higher in the healthy group than the group composed of chronic hbv patients and patients with spontaneous remission . since the methods used for kir genotyping permitted distinguishing groups of alleles of activating kir2ds4 , we found that there were no statistically significant differences for 2ds4 * 001 ( full - length exon 5 ) and 2ds4 * 003/4/6/7/8/9 ( deletion in exon 5 ) alleles between the 2 groups ( p>0.05 ) . additionally , there were no statistically significant differences between the rate of aa and bx genotypes of chronic hbv patients and patients with spontaneous remission and the control group ( p>0.05 ) . nk cells express multiple cell surface receptors , and during different infections different receptors are likely to be important . kirs have a variegated expression pattern , and their complex genetics indicate that they are involved in generating population diversity in antiviral immune response . previous studies have demonstrated several associations between various kir genes and/or their hla class i ligands in clinical progression of hbv infection [ 2022 ] and in the progression of this infection to hepatocellular carcinoma . these associations could be due to the capacity of interactions of hla class i molecules -especially hla - c and hla - b- with kirs and activation of nk cells . in the present study , we examined the genes encoding kir receptors and kir genotypes in chronic hbv patients , patients with spontaneous remission , and healthy controls in a turkish cohort . our results showed that the frequencies of both kir2dl3 and kir3ds1 genes were much higher in healthy controls than in chronic hbv patients and patients with spontaneous remission . this might indicate that the inhibitory kir2dl3 and activating kir3ds1 were possibly protector genes for hbv infection . similarly , gao et al , in their study comparing 182 chronic hbv patients with 140 healthy controls , reported that kir2dl3 : hla - c1 homozygosity was protective against hbv infection   and that kir2dl1 : hla - c2 was associated with susceptibility to hbv infection . in contrast , zhi - ming et al showed that kir2ds2 and kir2ds3 are hbv - susceptive genes , whereas kir2ds1 , kir3ds1 , and kir2dl5 may be protective genes that facilitate the clearance of hbv   in a han chinese population . population genetic analyses have revealed that the frequency and distribution of kir genes and haplotypes vary with ethnicity . although zhi - ming s study and ours were done in different populations , the kir3ds1 gene was found as a protector gene in both studies . in a wider study , lu et al analyzed kir genes in 150 patients with chronic hbv infection , 251 spontaneous resolvers , and 451 healthy controls . they found a lower frequency of the a haplotype and higher frequency of the b haplotype in patients exposed to hbv infection compared with healthy controls , implying a susceptibility effect of the b haplotype . in our study , there were no statistically significant differences between the rate of aa and bx genotypes of chronic hbv patients and patients with spontaneous remission and the control group . additionally , in a recent study , pan et al suggested the association of a combination of full - length form and 22 bp - deleted form of kir2ds4 ( kir2ds4/1d ) with hepatocellular carcinoma incidence in patients with chronic hbv . previous studies showed that there are important similarities between inhibitory and activating kir frequencies of patients with hepatitis c and hbv infections , despite these viruses being phylogenetically unrelated . khakoo et al reported that genes encoding the inhibitory nk cell receptor kir2dl3 and its ligand , hla - c1 , influence resolution of hepatitis c virus infection in caucasians and african americans with expected low infectious doses of hcv but not in those with high - dose exposure . chronic hbv patients , patients with spontaneous remission of hbv infection , and healthy controls in our population demonstrate that kir2dl3 and kir3ds1 are important immuno - genetic markers in determining antiviral immunity . to explore the role of kirs in hbv infections , the interrelation between kir genes , genotypes , and chronic hbv infection should be investigated in many different populations . models based on both activating and inhibitory receptors and their ligand interactions could be generated for detailed genetic studies in wider groups . thus , in order to confirm the present general results obtained in this study , functional studies and investigations in different populations are still needed to better understand this mechanism .