MISCELLANEOUS Different Strains of BK Polyomavirus: VP1 Sequences in a Group of Iranian Prostate Cancer Patients Maryam Vaezjalali1, 2, Helia Azimi1,3, Seyed Masoud Hosseini3, Afsoon Taghavi4, Hossein Goudarzi2* Purpose: BK virus (BKV) has a worldwide seroprevalence in humans. Based on sequences of the major capsid proteins, i.e. viral protein 1 (VP1), there are four BKV genotypes. Each genotype has its own subtypes, and was shown to be circulating independently in the human population. The aim of this study was to determine BKV genotypes and subtypes among Iranian patients with prostatic cancer, benign prostatic hyperplasia, and kidney transplantation. Materials and Methods: BKV DNA was extracted from prostatic cancers and benign prostatic hyperplasia blocks and also urine of kidney transplantation patients. BKV (VP1) gene was amplified partially (327nt) by homemade polymerase chain reactions and subjected for sequencing and phylogenetic analysis. Bioedit version 7.0 and Mega version 5.0 were used for sequence analysis and for comparing the results with world-driven BKV sequences. Results: All of BKV VP1 genes which were derived from Iranian patients were classified with subtype 1b2 strains from Germany and Turkey. Predicted amino acid sequences from the studied region of VP1 showed that all of these nucleotide diversities could change amino acid sequence numbers 60, 68, 72, 73 and 82 among VP1. Conclusion: The interesting point was that genetic analysis of derived sequences showed a different feature of genetic diversity among Iranian sequences. This feature has not been reported yet. This characteristic feature of Iranian BKV VP1 gene provides a unique cluster of sequences in phylogenetic tree. Keywords: BK virus; agnoprotein; genotype; prostate cancer; benign prostatic hyperplasia. INTRODUCTION BK virus (BKV), a member of Polyomaviridae, is ever-present among humans(1). In western coun- tries, BKV infects children asymptomatically(2,3). About 50% of children have BKV antibodies by four years old. By the age of 10 years old, 90-100% of them have seroconverted. BKV may persist in kidney tissue(4,5) and its contribution to human cancers is controversial. This virus has a double-stranded circular DNA genome of about 5 kbp packaged within a capsid of 45-50 nm in diameter(6). The BKV genome is divided into regu- latory, early, and late regions, and encodes five major proteins(7,8). The capsid contains three proteins: viral proteins (VP) 1, 2 and 3. VP1 gene is responsible for coding VP1, a major struc- tural protein that comprises approximately 80% of the total viral capsid protein. The VP1 protein has impor- tant domains which interact with viral receptors on host cells. A single amino acid change in VP1 protein can in- crease pathogenicity of mouse polyomavirus(9). Diversi- ties of VP1 gene lead to significant variation among BK viruses worldwide(10). BKV isolates are classified into four subtypes (I-IV) worldwide using serological and genotyping methods (1,11). Subtype I is prevalent throughout the world, sub- 1 Urology and Nephrology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2 Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 3 Department of Microbiology, Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran. 4 Department of Pathology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. *Correspondence: Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Velenjak St., Tehran, Iran. Postal Code: 1985717443. Tel: +98 21 23872556. Fax: +98 21 22439972. Mobile: +98 9126194134. E mail: Hossein.Goudarzi@sbmu.ac.ir. Received January 2017 & Accepted October 2017 type IV is in Asia and part of Europe, and subtypes II and III are rare in the world. These four genotypes cor- relate well with four serogroups, and were shown to be circulating independently in the human population. There are three reasons for researching about BKV genotypes and subtypes: 1) There is little information about the geographical circulation of BKV subtypes and subgroups. The effect of each genotype or subtype on clinical implications has previously been suggested (12,13); 2) Some researchers had reported that character- ization of BK virus' genetic mutations has biological and clinical implications(14). This data may be needed to follow potential relationships between BKV genotype and clinical disease in future; and 3) Diagnostic virol- ogy laboratories need the sequencing data of BKVs to ensure correct detection of all naturally occurring viral strains(15). This study tried to explain phylogenetic characteriza- tion of BKV strains among Iranian population and ana- lyze its different polymorphisms clinically. The BKV VP1 region was studied among Iranian patients with prostatic cancer, benign prostatic hyperplasia, and kid- ney transplantation. MATERIALS AND METHODS Twenty-five samples from our previous work on BKV Miscellaneus 44 were studied in this investigation(16). All patients were referred to a central hospital in Tehran. Each test was done considering one specimen as one patient. This study was approved by the Ethics Committee of the Urology and Nephrology Research Center (UNRC approval number 12-22 on 2012/3/12) and is in accord- ance with the Helsinki declaration of 1964. DNA extraction, gene amplification and sequencing DNA was extracted from urine samples with QIAamp® DNA Mini Kit (Qiagen, Hilden, Germany) and also from paraffin-embedded tissue blocks with QIAamp- DNA FFPE Tissue extraction kit (Qiagen, Duesseldorf, Germany). The protocols for working on the samples were followed based on our previous study (16). Briefly amplification of beta-globin was done for all samples to control DNA extraction. Then BKV amplification was done with specific primers(16). All experiments were done in a contamination-free environment. For each round of polymerase chain reaction (PCR), positive and negative controls were considered. PCR program included five minutes of denaturation at 94°C, followed by 35 rounds of an amplification cycle con- sisting of 35 seconds denaturation at 94°C, one minute annealing at 50°C for beta-globin and 55°C for BK vi- rus and one minute extension at 72°C, and a final ex- tension cycle of four minutes at 72°C. Beta-globin and BKV positive samples were detected with gel electro- phoresis and etidium bromide dying of 268 and 327 bp PCR product, respectively. One separate BK virus pos- itive specimen from the urine of infected patients was used as positive control. One beta-globin and all BKV PCR products were purified by QIAquick PCR purifi- cation kit (Qiagen, Duesseldorf, Germany). Then, they were subjected for direct sequencing (Genetic Analyzer ABI- 3130 DNA Sequencer, Fostercity, CA, USA) with 10 pmol of related PCR primers bi-directionally. Phylogenetic analysis Sequences derived from samples were read by Chro- mas software and aligned with reference sequences by Bioedit version 7. Their phylogenetic tree was drawn by Mega 5.0 software. The evolutionary history was inferred using the neighbor joining method. The boot- strap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the ana- lyzed taxa. The evolutionary distances were computed using the Kimura two-parameter method and were in the units of the number of base substitutions per site. A neighbor joining phylogenetic tree was reconstructed from the typing-region sequences obtained from Irani- an patients plus 88 reference sequences, using SA12 (a primate polyomavirus related to BKV) as the outgroup. RESULTS The patients from whom the samples were driven were from 10 provinces of Iran. All of them were men with a mean age of 68.0 ± 8.9 years old (range: 50 to 95 years old). Nephropathy of BKV was not reported. Also, derived sequences from one beta-globin PCR product confirmed the presence of beta-globin gene. Among 25 positive BKV samples, 15 samples were successfully sequenced. Nine samples were derived out from prostatic cancer tissues and five were derived out from benign prostatic hyperplasia tissues. Also, one sample was derived from a patient with kidney trans- plantation who was receiving immunosuppressive med- KP221578.1 KP221577.1 KP221579.1 KP221580.1 KP221581.1 KP221582.1 KP221583.1 KP221584.1 KP221585.1 KP221586.1 KP221587.1 KP221588.1 KP221589.1 KP221590.1 KP221591.1 AB276243.1- Turkey AB276242.1- Turkey 213493172- subtype1b2- China 213493180- subtype1b2- China KF468300.1- Germany KF468289.1- Germany JX195576.1- Germany 138996528- subtype1b2- Finland 213493186- subtype1b2- USA 213493184- subtype1b2- USA 213493192- subtype1b2- USA 213493188- subtype1b2- USA 118561556- subtype1b1- China 118561601- subtype1b1- China 118561595- subtype1b1- China 213493202- subtype1b1- USA 213493196- subtype1b1- USA 213493174- subtype1b1- USA 213493228- subtype1b1- USA 118561549- subtype1b1- China 118561615- subtype1b1- China 118561634- subtype1c- China 118561613- subtype1C- China 118561626- subtype1c- China 118561617- suype1c- China 118561632- subtype1c- China AB213286.1- subtype1c- china 118561558- subtype1c- China 118561636- subtype1c- China 118561575- subtype1c- China 118561565- subtype1c- China 118561624- subtype1c- China 118561622- subtype1c- China 118561607- subtype1c- China 118561593- subtype1c- China 118561580- subtype1c- China 118561603- subtype1c- China 83281137- subtype 1c- Japan 83281130- Subtype1c-Japan 83281158- subtype 1c-Japan 213493220- subtype1C- USA JN794032.1-Subtype 1a- France JN794016.1-Subtype 1a- France KC412484.1-Subtype 1a- Brazil KC412483.1-Subtype 1a- Brazil KC412462.1-Subtype 1a- Brazil 52839633- subtype4-Japan 118561554- subtype4- China 118561611- subtype4- China 118561597- subtype4- China 118561620- subtype4- China 109638401- subtype4- China 109638429- subtype4- Vietnam 213493226- subtype4- USA 138996952- subtype4- Philippines 118561630- subtype4-China 138996860- subtpye4- Myanmar 52839611- Subtype4- Japan 138997113- subtype4- Italia 118561628- sabtype4- China 118561609- subtype4- China 118561605- subtype4- China 213493222- subtype4- USA 213493194- subtype4- USA 213493190- subtype4- USA 213493176- subtype4- USA 83281144- subtype4-Japan 138996586- Subtype4- Greece 109638167- subtype4- Japan 52839547- subtype4- Japan 82524372-Simian virus 12 74 60 81 85 67 84 89 89 67 55 32 34 74 98 98 70 55 79 62 51 73 70 20 70 67 71 98 69 40 52 79 75 68 0.05 Figure 1. Phylogenetic tree of BKV VP1- gene partial sequence constructed using 15 isolates from this study along with Genbank reference sequences. SA12 (a primate polyomavirus related to BKV) was considered as the outgroup. BK virus genotypes among Iranian patients-Vaezjalali et al. Vol 15 No 02 March-April 2018 45 ications. In the resulted tree (Figure 1), typing-region sequences were divided into clusters corresponding to subtype I and all of Iranian BKV strains were clustered with sub- type 1b2 strains from Germany, Turkey and USA. It was apparent that Iranian BKV sequences are classified as a different cluster among other subtype 1b2 strains. The sequences derived from this study have been sub- mitted to GenBank by accession numbers: KP221577- KP221591. In this study nucleotides 118-393 and therefore predi- cated amino acid 40-132 of VP1 were investigated. Ira- nian strains alignment with reference sequence (acces- sion number: JX195576.1) showed that five nucleotides were different among the derived VP1 region, 180, 202, 215, 218 and 244. The predicted amino acid sequenc- es from the studied region of VP1 showed that all of these nucleotide diversities could change amino acid sequence numbers 60, 68, 72, 73 and 82 among VP1. DISCUSSION In this study, phylogenetic analysis was done on 327 nucleotides of BKV VP1 region derived out from 15 patients. There were two remarkable findings. One was that phylogenetic analysis of these sequences es- tablished one cluster for designated Iranian sequences. This cluster was classified with high bootstrap value with BKV sequences subtype 1b2 from Turkey and Germany. Subtype 1b2 is of type 1 which had been re- ported in Europe previously(15). Subtypes a, b1 and c are other subgroups of subtype 1 which are most prevalent in Africa, South-east Asia and North-east Asia, respec- tively(17-20). Two previous studies in Iran had detected BKV subtypes among kidney transplant recipients by restriction fragment length polymorphism - polymerase chain reaction (RFLP-PCR). In 2012, researchers reported BKV subtype 1 among 12 Iranian-Azeri people from north-west of Iran(12). In 2015, researchers reported BKV subtype 1 from east of Iran(21). From 51 BKV samples in that study, 94.11% were subtype I and 5.89% were subtype IV using the RFLP method. None of the patients' urine samples were positive for subtypes II and III. One reason for achiev- ing different results in our study might be because of fewer samples, ethnicity or different clinical samples. However, sequencing is the standard test for genotyp- ing which can help researchers analyze viral sequences sufficiently. On the other hand, in a new study, BKV subtype 1b2 was reported among Iranian HIV-infected patients(22). The other significant point was that our studied se- quences were obtained from different clinical sources and geographical regions of Iran. Despite controlling PCR conditions and preventing DNA contamination, no difference was observed between strains derived from patients with prostate cancer, benign prostatic hyperpla- sia and kidney transplantation. All of the derived se- quences belonged to BKV subtype 1 in this study. Pre- vious studies had reported that despite different clinical samples, subtype 1 was predominant around the world (23-25). Since BKV subtype I was geographically prevalent, some researchers had suggested that there is no signifi- cant correlation between BKV subtypes and geographi- cal regions(17). However, it seems that different research design of studies might be the cause of this theory. Pre- vious studies had reported 287 bp of VP1 as a remark- able region for BKV genotyping. Two articles reported that full genome sequencing can detect viral genotypes more powerfully with 5% higher probability for boot- strap subgroup classification(26,27). However, these two reports asserted that the 287 bp typing region is useful to classify isolates into subtypes I to IV. Totally, sequencing of the whole VP1 region or first part of the LTag is required to classify isolates into subgroup Ib-1 or Ib-2, supporting findings in previous reports(14,27). Zheng and colleagues analyzed 30 typ- ing regions and also full genome sequencing of BKVs derived out from different geographical areas of the world(19). They reported that the phylogenetic analysis based on complete DNA sequences supports not only the subtype classification of BKV isolates, but also the sub-classification of subtype I isolates. In addition, this phylogenetic analysis allowed sub-classification of sub- type IV into its subgroups. Also, they suggested that host-linked evolution is the general mode of polyoma- virus (JC and BK viruses) evolution(19). Additionally, their results indicated certain unique aspects of the re- lationship between BKV and humans. This was a the- ory for tracing the geographical origins of unidentified cadaver based on BK genotype in a report from Ikegaya and colleagues(28). Zhong and colleagues(20) studied BKV genotypes of American, European, and Asian populations. They re- ported the highest frequency of subtype I in all pop- ulations compared to subtype IV which was variable among populations. Subgroup I/c was prevalent in native Japanese but was rare in the second generation of this population(19,20). This observation can also be reconciled with the co-migration hypothesis. This hy- pothesis assumes that the children of migrant Japanese have acquired BKV infection not from their parents, but from European Americans living in their local commu- nity(19,20). Therefore, it seems that prevalence of BKV subgroups among a geographical region might not be related to ethnicity. By aligning the derived sequences with the reference se- quence (accession number: JX195576), we found VP1 gene mutations which were different from other reports. All amino acid variations in this study (positions 60, 68, 72, 73 and 82) localize to the BC loop. On the oth- er hand, subtypes are determined by the VP1 sequence between amino acids 61 and 83, which is the variable antigenic region and maps to the BC1 and BC2 loops. (29) Therefore, it was apparent that the variations were not randomly distributed but seemed to be arranged in “VP1 hotspots.” Amino acids at positions 60, 68, 72, 73 and 82 in the VP1 protein showed an interesting pat- tern of changes(14). Some researchers had suggested that amino acid substitutions at these five locations might result in type-determining changes in three-dimensional protein configurations(14). Since the BC loop is believed to interact with the cellular receptor of BKV, it would be speculated that the genotype specific amino acid changes might alter BKV tissue tropism(14). However, the finding of Pastrana and colleagues proved that dif- ferent BKV genotypes have different cellular tropisms and pathogenic potentials in vivo(30). Recently, two published data have pointed that like RNA, BKV viruses are able to produce quasispecies. A study had found that sequencing of BKV isolates sub- cloned from BKV in nephropathy patients revealed a high percentage of variants in the urine (40%) in the VP1 subtyping region(31). In vitro analysis of several vi- BK virus genotypes among Iranian patients-Vaezjalali et al. Miscellaneus 46 ral variants revealed that all variants which recovered from the urine of BKV associated nephropathy patients produced infectious viral particles. Studied BKVs were replication-competent in cell culture while some of the variants induced cytopathic changes in infected cells compared to the major BKV subtype and VP1 subtype I. These results suggest that rare BKV VP1 variants are more frequently associated with disease and that some variants could be more cytopathic than others in kidney transplant recipients(31). No phylogenetic analysis reports of BKV genome were found among patients with prostate cancers and benign prostatic hyperplasia tissues. In our study 93% of stud- ied samples were derived out from patients with pros- tate cancers and benign prostatic hyperplasia tissues. This remarkable VP1 gene diversity of BKV may have an implication role for clinical diagnostics. BKV can cause malignancy in animal models. Still conclusive evidence is lacking regarding a causal connection be- tween BKVs and human cancer(30). Our study had some limitations. We used relatively few samples. Therefore, the results cannot be generalized to the Iranian population. Nevertheless, we consist- ently reported five new mutations in all studied patients which were not previously reported in Iranian patients which add to the value of our findings. Also as stated above we had no information regarding the serological tests of BKV infection in our patients. CONCLUSIONS Phylogenetic analysis of the VP1 gene sequences in our study supports the existence of genotypes 1b2 in the studied sample of Iranian patients in this study. Finding BKV subtype 1 in Iran was consistent with other Ira- nian studies, which had reported BKV among Iranian kidney transplanted patients. Our data documented the phylogenetic diversity of Iranian BKV and established the existence of clades not previously recognized in the literature. In this study BKV sequence of Iranian strains showed five mutations within the VP1 (positions 60, 68, 72, 73 and 82) which distinguished these strains from other type 1b2 sequences. Iranian BKV strains do not form distinct clusters from each other. However, they were classified as a unique cluster among 1b2 sub- type sequences. CONFLICT OF INTEREST None declared. ACKNOWLEGEMENT This study has been supported by Urology and Neph- rology Research Center, Shahid Beheshti University of Medical Sciences. The authors would like to thank Seyed Muhammed Hussein Mousavinasab for his sin- cere cooperation in editing this text. REFERENCES 1. Knowles WA. Propagation and assay of BK virus. Methods Mol Biol. 2001;165:19-31. 2. Jozpanahi M, Ramezani A, Ossareh S, et al. BK Viremia among Iranian Renal Transplant Candidates. Iran J Pathol. 2016;11:210-5. 3. Konietzny R, Fischer R, Ternette N, et al. Detection of BK virus in urine from renal transplant subjects by mass spectrometry. 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BK virus genotypes among Iranian patients-Vaezjalali et al.