bk virus ( bkv ) belongs to the human polyomaviridae , initially isolated from urine sample of a 29-year - old male patient with renal blockage and failure at 1971 .
bk virus is an abbreviation of the name of the first patient whom the virus was isolated from .
usually , primary infection is occurred during childhood then the virus could be latent through life , especially in the kidneys and urinary system ( 1 ) .
it became reactive after immunocompromisation , status such as pregnancy or transplantation ; in such patients bkv develops bkv - related renal failure and nephropathy .
it is reported that bkv became reactivated in 10 - 60% of the cases of renal transplant patients from which 1 - 5% would undergo nephropathy ; half of the patients with nephropathy reject their transplanted kidney .
risk factors of bkv - induced nephropathy are not well - known . however , immunosuppressant drugs , transplantation and bkv itself account as the major risk factors ( 2 - 5 ) . isolated bkv from different locations of the world are grouped into four subtypes using serological and genotyping methods .
subtype i is the dominant one and has worldwide distribution ; subtype iv is the more frequent after subtype i and mostly isolated from east asia .
although subtypes ii and iii are isolated worldwide , their frequencies are low . furthermore , according to the phylogenic investigations subtype i itself is divided into 4 subgroups including subgroups 1/a , 1/b-1 , 1/b2 and 1/c ; each one is distributed in a certain geographical location .
subtype iv , itself is divided into six subgroups in phylogenic studies including 4/a-1 , 4/a-2 , 4/b-1 , 4/b-2 , 4/c-1 and 4/c-2 ( 6 , 7 ) . according to previous reports
, there are no clear correlation between bkv subtype determination and clinical outcome ; thus , our study will have epidemiological benefits .
according to our knowledge there are no data about bkv prevalence and its genotypes in southwest of iran . considering the high prevalence of renal failure and kidney transplant patients in this part and the role of bkv in graft rejection , this study aimed to determine the prevalence of bkv infection in renal transplant recipients referred to golestan hospital in ahvaz , iran .
therefore , the results of our study and setting up of bkv detection can be helpful for physicians to recognize at risk patients .
urine samples of 122 kidney transplant patients were gathered from ahvaz golestan hospital , southwest of iran , during 2010 - 2011 .
all of our cases were immunocompromised renal transplant patients and there were no specific inclusion and exclusion criteria .
a 50 ml urine specimen was added to 0.5 ml of 0.5 mol edta with ph = 8 ; all samples were sending to the infectious and tropical diseases research center of ahvaz jundishapur university of medical sciences .
each sample was centrifuged in 1500 rpm and 4c for 10 minutes ; the pellet kept in -20c till test running time .
viral dna was extracted using roche high pure viral nucleic acid extraction kit ( ref .
primers used to amplify the typing region were 327 - 1 pst 5'- gcc tgc agc aag tgc caa aac tac taa t-3 ' and 327 - 2 hin 5'- gca agc ttg cat gaa ggt taa gca tgc -3 ' underlined nucleotides were added to create a psti or hindiii cleavage site ( 8) . extracted dna was amplified using 5 l dna template , 0.3 l taq polymerase , 1 l dntp , 0.25 l each forward and reveres primers , 2 l mgcl2 and 5 l 10x pcr buffer , all mixed in a microtube and the ultimate volume reach to 50 l using nuclease - free distilled water . for each run , we used diethylpyrocarbonate ( depc ) water as a negative control and confirmed positive sample depc water as a negative control and confirmed positive sample from our previous study ( 9 ) as a positive control .
after activation of reaction in 95c for 5 minutes , amplification was performed for 50 cycles .
cycle program was as : 94c for 60 , 55c for 60 and 72c for 90 seconds .
polymerase chain reactions ( pcrs ) were performed using the peqlab thermocycler ( peq star , 96 universal gradient -germany ) .
the 342 bp amplification product was detected using 1.7% gel agarose socked in ethidium bromide ( figure 1 ) .
sequences were analyzed using the neighbor toning phylogenetic tree software and compared by reference ones .
p , positive control ; n , negative control ; l , 100 bp ; 1 - 4 , positive samples ; 5 - 6 , negative samples a 342 bp which contained a 287 bp bk type specific fragment , and which was amplified with pcr , treated by the restriction enzymes cfri13 i , alu i and rsa i according to their package insert instructions . table 1 shows fragment lengths resulted from restriction enzymes action on bk subtypes .
one to 3 l pcr product was added to 1 - 10 units of each enzymes , incubated in 37c for 1 hour ( if necessary overnight ) . then digested products were separated using 3% agarose gel electrophoresis .
urine samples of 122 kidney transplant patients were gathered from ahvaz golestan hospital , southwest of iran , during 2010 - 2011 .
all of our cases were immunocompromised renal transplant patients and there were no specific inclusion and exclusion criteria .
a 50 ml urine specimen was added to 0.5 ml of 0.5 mol edta with ph = 8 ; all samples were sending to the infectious and tropical diseases research center of ahvaz jundishapur university of medical sciences .
each sample was centrifuged in 1500 rpm and 4c for 10 minutes ; the pellet kept in -20c till test running time .
viral dna was extracted using roche high pure viral nucleic acid extraction kit ( ref .
primers used to amplify the typing region were 327 - 1 pst 5'- gcc tgc agc aag tgc caa aac tac taa t-3 ' and 327 - 2 hin 5'- gca agc ttg cat gaa ggt taa gca tgc -3 ' underlined nucleotides were added to create a psti or hindiii cleavage site ( 8) . extracted dna was amplified using 5 l dna template , 0.3 l taq polymerase , 1 l dntp , 0.25 l each forward and reveres primers , 2 l mgcl2 and 5 l 10x pcr buffer , all mixed in a microtube and the ultimate volume reach to 50 l using nuclease - free distilled water . for each run , we used diethylpyrocarbonate ( depc ) water as a negative control and confirmed positive sample depc water as a negative control and confirmed positive sample from our previous study ( 9 ) as a positive control .
after activation of reaction in 95c for 5 minutes , amplification was performed for 50 cycles .
cycle program was as : 94c for 60 , 55c for 60 and 72c for 90 seconds . a final extension 72c for 5 minutes was the ultimate step of cycles .
polymerase chain reactions ( pcrs ) were performed using the peqlab thermocycler ( peq star , 96 universal gradient -germany ) .
the 342 bp amplification product was detected using 1.7% gel agarose socked in ethidium bromide ( figure 1 ) .
sequences were analyzed using the neighbor toning phylogenetic tree software and compared by reference ones .
p , positive control ; n , negative control ; l , 100 bp ; 1 - 4 , positive samples ; 5 - 6 , negative samples
a 342 bp which contained a 287 bp bk type specific fragment , and which was amplified with pcr , treated by the restriction enzymes cfri13 i , alu i and rsa i according to their package insert instructions . table 1 shows fragment lengths resulted from restriction enzymes action on bk subtypes .
one to 3 l pcr product was added to 1 - 10 units of each enzymes , incubated in 37c for 1 hour ( if necessary overnight ) . then digested products were separated using 3% agarose gel electrophoresis .
in this study , a total of 122 patients with kidney transplantation were admitted to golestan hospital in ahvaz , southwest of iran , for the determination of bkv infection using pcr . considering that our study aimed to determine the epidemiological distribution of bkv
patients were from both sexes including 76 ( 62.3% ) males and 46 ( 37.7% ) females and also aged between 13 - 86 years ( mean 38.6 ) . from all study population , 51 ( 41.8% ) of urine samples
frothy - eight cases ( 94.11% ) were subtype i and iii others ( 5.89% ) were subtype iv using the rflp method .
sequencing of positive samples confirmed the obtained results , which were shown that bk subtypes were positive for subtypes i and iv and any case was positive for subtypes ii and iii .
a frequency of 42% is reported for bkv in chinese immunocompetent individuals ( 10 ) . according to the current reports
, infected donors could transport the virus to the recipients ( 11 , 12 ) .
the bkv isolates worldwide are classified into four subtypes ( i - iv ) using serological and genotyping methods ( 4 ) .
subtype i is widespread throughout the world , subtype iv shows a geographical distribution biased for east asia , and subtypes ii and iii are rarely detected throughout the world ( 6 , 7 ) . in the current study , we have reported 41.8% frequency of the virus in our study population which is much higher than that of reported by bohl et al .
( 13 ) that were about 8% and 13% . in a previous study , which was conducted by the author and colleagues in 1386 , the prevalence of the virus in the urine of patients , were reported 38% ( 9 ) .
chen and coworkers have reported 20%-30% frequency rates of bk - positive urines of south asia immunecompromised individuals ( 14 ) .
our results show that the most frequent subtypes are subtype i and iv of bkv , respectively which is in accordance with other reports from other parts of world and from iran .
such result proposes that till now the subtype i is the most frequent subtype worldwide .
( 6 ) have declared that the origin of subtype iv is from asia and is founded in variable rates in asian populations . in turn
have reported that the most frequent bkv subtype in germanic patients is a subtype i ( 15 ) .
have evaluated the prevalence of bkv in japanese recipients of kidney or bone marrow transplantations ; their results showed that the frequency rates of subtypes i , iii and iv were 10%-20% , 70%-80% and 2%-3% , respectively ( 8) .
have reported that the most frequent bkv subtype is a subtype i in different populations of europeans ( 16 ) . however , the prevalence of subtype iv was variable in different studies .
chen et al . ( 14 ) study in south - east of asia showed that in japan and south - east parts of china , subtype i was more frequent than subtype iv ; in north - east of china and vietnam , subtypes i and iv had nearly equal frequencies but in mongolian population subtype iv was the most frequent one ( 16 ) .
however , some epidemiologic investigations deny any significant dependency of bkv frequency to geographic distribution ( 15 ) .
the dependency of bkv frequency to geographical distribution is considerable about subtype iv and not considerable about subtype i , which is the most frequent subtype worldwide ( 8) . some studies have divided subtypes into subgroups and their population dependent frequencies are evaluated ( 8 , 14 , 15 , 17 , 18 ) . furthermore , motazakker and collages have isolated only subtype i using rflp from uromia kidney transplant patients ( 19 ) . according to the above - mentioned reports , although the patients with kidney transplantation who had bkv were more at risk than bkv negative patients , the different bkv subtypes did nt affect the clinical outcomes in those patients .
when virus has reactivated , it spread by cell - to - cell ( 20 - 22 ) . in some conditions that the immune system is out of control and the patients
lysis of infected tubule cells results in viral leakage into the tubule lumen and urine .
the spread of viral infection leads to necrosis of renal cells and its clinical and laboratory manifestations .
our work is the second study in iran and considering huge numbers of transplantation in iran and khuzestan province , southwestern of iran , in addition to the role of this virus in kidney transplant rejection , a routine evaluation of bk positivity is recommended both for graft recipients and donors .
 background : bk virus ( bkv ) belongs to the human polyomaviridae and the primary bkv infection is occurred during childhood then the virus could be latent through life , especially in the kidneys and urinary system . 
 it became reactive after an immunocompromised status , such as pregnancy or transplantation . 
 isolated bkv from different locations of the world is grouped into four subtypes using serological and genotyping methods . 
 the bkv subtype i is the dominant one and has worldwide distribution.objectives:according to our knowledge , there are no data about the bkv prevalence and its genotypes in southwest part of iran . considering the high prevalence of renal failure and kidney transplant patients in this part , and the role of bkv in graft rejection , this study aimed to determine the prevalence of bkv infection in renal transplant recipients referred to golestan hospital in ahvaz city , iran.patients and methods : urine samples were collected from 122 kidney transplant recipients referred to golestan hospital in ahvaz , southwest of iran . 
 the extracted dna was amplified by polymerase chain reaction , and subtype of each positive sample was determined using restriction fragment length polymorphism ( rflp ) and sequencing methods.results;from all study population , 51/122 ( 41.8% ) urine samples were positive for bkv dna and the other samples were negative ( 71/122 ) . 
 forty - eight cases ( 94.11% ) were subtype i and 3 others ( 5.89% ) were subtype iv using the rflp method . 
 none of the patient s urine samples were positive for subtypes ii and iii.conclusions:our work is the second study in iran and considering huge numbers of transplantation in iran and khuzestan province , south western of iran , in addition to the role of this virus in kidney transplant rejection , routine evaluation of bkv positivity is recommended both for graft recipient and donors . 
 this helps better transplantation result and may prevent graft rejection . 
1. Background
2. Objectives
3. Patients and Methods
3.1. Patients
3.2. Sample Collection, DNA Extraction and Polymerase Chain Reaction
3.3. Restriction Fragment Length Polymorphism
4. Results
5. Discussion