The prostate gland is one of the most essential male accessory glands. This gland is susceptible to various pathological conditions, among which both malignant and benign conditions are the most common [1Verhamme KM, Dieleman JP, Bleumink GS, et al. Triumph Pan European Expert Panel. Incidence and prevalence of lower urinary tract symptoms suggestive of benign prostatic hyperplasia in primary care--the Triumph project. Eur Urol 2002; 42(4): 323-8.
[http://dx.doi.org/10.1016/S0302-2838(02)00354-8] [PMID: 12361895] ]. Benign Prostatic Hyperplasia (BPH), which is not a cancer, is common among older men and occurs when the prostate gland is enlarged. When the gland becomes larger it ‘squeezes’ the urethra, thus causing several complications, such as difficulty urinating and frequent urination during the day [2Grossman DC, Curry SJ, Owens DK, et al. US Preventive Services Task Force. Screening for Prostate Cancer: US Preventive Services Task Force Recommendation Statement. JAMA 2018; 319(18): 1901-13.
[http://dx.doi.org/10.1001/jama.2018.3710] [PMID: 29801017] ]. PCa is a biologically heterogeneous tumor and is one of the leading causes of cancer death in men [3Rodríguez H, Levican J, Muñoz JP, et al. Viral infections in prostate carcinomas in Chilean patients. Infect Agent Cancer 2015; 10: 27.
[http://dx.doi.org/10.1186/s13027-015-0024-y] [PMID: 26330890] ]. 241,740 new cases and 28,170 deaths were estimated in 2012 [4Delbue S, Matei DV, Carloni C, et al. Evidence supporting the association of polyomavirus BK genome with prostate cancer. Med Microbiol Immunol (Berl) 2013; 202(6): 425-30.
[http://dx.doi.org/10.1007/s00430-013-0304-3] [PMID: 23821367] ]. It is the second leading cause of cancer-related death in men in the United States [5Elimam ME, Abdelraof S. Incidence of carcinoma of the prostate in patients with normal prostatic specific antigen following prostatectomy. Glob J Medi Res 2013; 13: 26-35.]. The underlying causes of PCa are not fully understood, but it is likely to occur due to a combination of factors, such as aging, family history and dietary factors in addition to infectious agents [4Delbue S, Matei DV, Carloni C, et al. Evidence supporting the association of polyomavirus BK genome with prostate cancer. Med Microbiol Immunol (Berl) 2013; 202(6): 425-30.
[http://dx.doi.org/10.1007/s00430-013-0304-3] [PMID: 23821367] -6Provenzano M, Keller P. The potential therapeutic usefulness of targeting BK polyoma virus in prostate cancer. Chemo Open Access 2015; 5: 1.]. Pooled data from population studies on PCa risk have reported an 80% increase in the risk of PCA in men with a history of prostatitis, although the detection of potential bias plays a role in it. Proliferative Inflammatory Atrophy (PIA) is common in normal and cancerous prostate and is a regenerative lesion after injury or trauma. It has been suggested that PIA is a precursor lesion of PCa and several genetic mutations have been identified in PIA, including glutathione S-transferase pi 1 (GSTP1) hypermethylation, p53 mutations, and alterations on chromosome 8 [7Martinez-Fierro ML, Leach RJ, Gomez-Guerra LS, et al. Identification of viral infections in the prostate and evaluation of their association with cancer. BMC Cancer 2010; 10: 326.
[http://dx.doi.org/10.1186/1471-2407-10-326] [PMID: 20576103] ].

Several pieces of evidence support an infectious pathway in PCa development. In particular, Sexually Transmitted Infections (STIs) have been implicated in PCa etiology in many studies. A meta-analysis of 29 case-control studies found an increased relative risk of PCa in men with a history of any STI with an odds ratio of 1.5 and 95% Confidence Interval (CI), (1.3-1.7). Several sexually transmitted organisms have been detected in the prostate, including Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Treponema pallidum, human papillomavirus, herpes simplex virus, and human herpesvirus type 8. Serum antibodies against herpes simplex virus and Trichomonas vaginalis in men have been associated with PCa risk, whereas the presence of the DNA of human papillomavirus in prostate tissues has also been associated with the risk of PCa [8Wright JL, Lin DW, Stanford JL. Circumcision and the risk of prostate cancer. Cancer 2012; 118(18): 4437-43.
[http://dx.doi.org/10.1002/cncr.26653] [PMID: 22411189] ]. Infectious agents may lead to cancer by several potential mechanisms. Although, direct cellular transformation can occur by viruses, several changes in the tissue microenvironment can occur if a chronic inflammatory state is reached after the infection. These include damage due to reactive oxygen species along with cytokine-induced angiogenesis and cellular proliferation, all of which may be involved in carcinogenesis [9De Marzo AM, Platz EA, Sutcliffe S, et al. Inflammation in prostate carcinogenesis. Nat Rev Cancer 2007; 7(4): 256-69.
[http://dx.doi.org/10.1038/nrc2090] [PMID: 17384581] ].

The physiopathology of BKV-induced inflammation and the role of a cofactor in prostate and other cancers can be summarized by the fact that BKV T antigens, such as JCV, display multiple functions which alter the normal physiological metabolism of cells, ultimately leading to immortalization and neoplastic transformation. The main property of Tag in relation to the transformation and oncogenicity is its ability to bind and block the functions of tumor suppressor proteins p53 and pRB family (p105 RB1, p107 and p130 RB2). BKV Tag also induces numerical and structural chromosomal alterations in human embryonic fibroblasts, characterized by gaps, breaks, dicentric and ring chromosomes, deletions, duplications and translocations. Chromosome damage in human cells transfected with BKV early region was evident before the appearance of immortalization and the morphologically transformed phenotype [10Das D, Wojno K, Imperiale MJ. BK virus as a cofactor in the etiology of prostate cancer in its early stages. J Virol 2008; 82(6): 2705-14.
[http://dx.doi.org/10.1128/JVI.02461-07] [PMID: 18160432] -13Zambrano A, Kalantari M, Simoneau A, Jensen JL, Villarreal LP. Detection of human polyomaviruses and papillomaviruses in prostatic tissue reveals the prostate as a habitat for multiple viral infections. Prostate 2002; 53(4): 263-76.
[http://dx.doi.org/10.1002/pros.10157] [PMID: 12430138] ].

The human BK polyomavirus is a member of the polyomavirus family. The virus has an icosahedral shaped capsid which is unenveloped, measuring about 40-50 nm. The capsid encloses a circular double-stranded DNA genome of approximately 5100 nucleotides coated by host-cell histones [14Gardner SD, Field AM, Coleman DV, Hulme B. New human papovavirus (B.K.) isolated from urine after renal transplantation. Lancet 1971; 1(7712): 1253-7.
[http://dx.doi.org/10.1016/S0140-6736(71)91776-4] [PMID: 4104714] ]. The virus was first isolated from the urine of a renal transplant patient in 1971 [14Gardner SD, Field AM, Coleman DV, Hulme B. New human papovavirus (B.K.) isolated from urine after renal transplantation. Lancet 1971; 1(7712): 1253-7.
[http://dx.doi.org/10.1016/S0140-6736(71)91776-4] [PMID: 4104714] ] and infects almost 90% of the human population worldwide. It rarely causes diseases in immune-competent individuals and resides in a subclinical persistent state in the urinary tract of healthy individuals and reactivates in immunosuppressed transplant patients, in whom it is associated with certain diseases, such as hemorrhagic cystitis and polyomavirus nephropathy [15Tognon M, Corallini A, Martini F, Negrini M, Barbanti-Brodano G. Oncogenic transformation by BK virus and association with human tumors. Oncogene 2003; 22(33): 5192-200.
[http://dx.doi.org/10.1038/sj.onc.1206550] [PMID: 12910256] ]. Urinary shedding has been reported to occur asymptomatically and intermittently in healthy individuals [16Dalianis T, Hirsch HH. Human polyomaviruses in disease and cancer. Virology 2013; 437(2): 63-72.
[http://dx.doi.org/10.1016/j.virol.2012.12.015] [PMID: 23357733] ].

The genome of BKV is divided into two transcriptional regions called early and late regions in addition to the third non-transcriptional region called the regulatory region. The genome of a typical BK polyomavirus codes for between 5 and 9 proteins, two from the early region and the rest from the late region. The early region is transcripted by the host cell's RNA polymerase II and codes for two proteins, the small and large tumor antigens, produced by alternative splicing [17Ahuja D, Sáenz-Robles MT, Pipas JM. SV40 large T antigen targets multiple cellular pathways to elicit cellular transformation. Oncogene 2005; 24(52): 7729-45.
[http://dx.doi.org/10.1038/sj.onc.1209046] [PMID: 16299533] ]. The Large Tumor Antigen (LTAg) is expressed early in the infectious cycle and is essential for virus proliferation. It is about 600-800 amino acids in length containing four well-conserved protein domains as well as several intrinsically disordered regions. The antigen has two primary functions, both related to virus replication. Its first function is to unwind the viral DNA and prepare it for replication, while the second is to destroy the cell cycle by interaction with the host cell protein. In addition, the LTAg promotes cellular transformation by interfering with the tumor suppressor functions of Rb, p107, p130, and p53 (Fig. 1) [17Ahuja D, Sáenz-Robles MT, Pipas JM. SV40 large T antigen targets multiple cellular pathways to elicit cellular transformation. Oncogene 2005; 24(52): 7729-45.
[http://dx.doi.org/10.1038/sj.onc.1209046] [PMID: 16299533] -19Maurizio P, Etienne XK. The potential therapeutic usefulness of targeting BK Polyomavirusin prostate cancer. Chemo Open Access 2015; 5: 1-2.]. Another protein that is expressed early in the BKV infectious cycle is the small Tumor Antigen (TAg). Unlike the LTAg, it is not essential for viral replication, instead it induces tumorogenesis and promotes anchorage-independent growth of transformed cells by interaction with the cell protein phosphatase 2A (Fig. 1) [18Yu J, Boyapati A, Rundell K. Critical role for SV40 small-t antigen in human cell transformation. Virology 2001; 290(2): 192-8.
[http://dx.doi.org/10.1006/viro.2001.1204] [PMID: 11883184] , 19Maurizio P, Etienne XK. The potential therapeutic usefulness of targeting BK Polyomavirusin prostate cancer. Chemo Open Access 2015; 5: 1-2.]

Fig. (1) Genome structure of BKV. The transcription of both early and late coding regions proceeds in a bidirectional way from the Origin of Replication (ORI) within the Noncoding Control Region (NCCR). The transcriptional splicing regions are represented by dashed lines. The late coding regions encode structural proteins (VP1, VP2, and VP3), while the early coding regions transcribe the tumorigenic proteins (LTA and STA). The late coding regions also encode the non-immunogenic Agno protein. The expression of miRNA complementary to the 3= end of LTA has been shown to be involved in replication control of BKV (36).

The first genotyping schema for BKV described by Jin et al. in 1993 was based on a very short segment of the VP1 gene (nucleotides 1744 to 1812). However, phylogenetic trees based on LTA sequences were introduced and validated recently [20Luo C, Bueno M, Kant J, Martinson J, Randhawa P. Genotyping schemes for polyomavirus BK, using gene-specific phylogenetic trees and single nucleotide polymorphism analysis. J Virol 2009; 83(5): 2285-97.
[http://dx.doi.org/10.1128/JVI.02180-08] [PMID: 19109389] ]. The International Committee on Taxonomy of Viruses currently classifies polyomaviruses primarily according to the sequence identity of their LTag genes. This system has been questioned by phylogenetic studies suggesting that the evolutionary history of LTag and major capsid protein VP1 is divergent and that some modern polyomaviruses represent chimeric lineages [18Yu J, Boyapati A, Rundell K. Critical role for SV40 small-t antigen in human cell transformation. Virology 2001; 290(2): 192-8.
[http://dx.doi.org/10.1006/viro.2001.1204] [PMID: 11883184] , 19Maurizio P, Etienne XK. The potential therapeutic usefulness of targeting BK Polyomavirusin prostate cancer. Chemo Open Access 2015; 5: 1-2.]. Based on the serological and genotyping method, the BKV isolated from various locations of the world is categorized into four subtypes [21Kaydani GA, Makvandi M, Samarbafzadeh A, et al. Prevalence and distribution of BK virus subtypes in renal transplant recipients referred to golestan hospital in ahvaz, Iran. Jundishapur J Microbiol 2015; 21(8): e16738.]. Subtype I is the most dominant and has a worldwide distribution, followed by subtype IV which is mostly isolated from East Asia. However, subtypes II and III have low frequencies compared to the others [22Andi K, Olaf R, Bininda E, Peter W, Roland Z. Evolution of four BK virus subtypes, infection, genetics and evolution. Infect Genet Evol 2008; 8: 632-43.
[http://dx.doi.org/10.1016/j.meegid.2008.05.006] ]. In addition, the phylogenic investigations revealed that there were four subgroups of subtype I, subgroups 1/a, 1/b-1, 1/b2 and 1/c. While, Subtype IV is divided into six subgroups, 4/a-1, 4/a-2, 4/b-1, 4/b-2, 4/c-1 and 4/c-2 [21Kaydani GA, Makvandi M, Samarbafzadeh A, et al. Prevalence and distribution of BK virus subtypes in renal transplant recipients referred to golestan hospital in ahvaz, Iran. Jundishapur J Microbiol 2015; 21(8): e16738.].

In Sudan, the relationship between viruses and cancer has been studied in only a limited studies [23Gorish BM. Frequency of HBV among hepatocellular carcinoma patients in khartoum state, sudan. J Carcinog Mutagen 2018; 9: 320., 24Dafalla AB, Elnil YF, Gorish BM. Seroprevalence of cytomegalovirus infection among leukemic patients in khartoum state. Virol Mycol 2018; 7: 183.]. In our previous study, the existence of the relationship between BKV and prostate cancer was determined [25Gorish BMT, Ournasseir MEH, Shammat IM. A correlation study of BK Polyoma Virus infection and prostate cancer among sudanese patients - immunofluorescence and molecular based case-control study. Infect Agent Cancer 2019; 14: 25.
[http://dx.doi.org/10.1186/s13027-019-0244-7] [PMID: 31548852] ] and the molecular characterization of BKV was described because only a few researchers have addressed BKV Subtypes.However, most previous studies have focused only on the isolates related to BKV nephropathy. The effect of each subtype on the clinical implications had previously been highlighted by various researchers [26Motazakker M, Bagheri M, Imani M. Subtyping of BK Virus in iranian turkish renal transplant recipients by RFLP-PCR. Maedica (Buchar) 2012; 7(1): 10-3.
[PMID: 23118813] , 27Wang ZY, Hong WL, Zhu ZH, et al. Phylogenetic reconstruction and polymorphism analysis of BK virus VP2 gene isolated from renal transplant recipients in China. Exp Ther Med 2015; 10(5): 1759-67.
[http://dx.doi.org/10.3892/etm.2015.2723] [PMID: 26640547] ]. Some researchers have reported that the characterization of the genetic mutations of BK virus ge may have biological and clinical effects [28Sharma PM, Gupta G, Vats A, Shapiro R, Randhawa P. Phylogenetic analysis of polyomavirus BK sequences. J Virol 2006; 80(18): 8869-79.
[http://dx.doi.org/10.1128/JVI.00510-06] [PMID: 16940499] ]. Thus, our study attempted to determine the phylogenetic characterization of BKV strains among Sudanese patients with a prostate disorder, such as prostate cancer and benign prostatic hyperplasia.

This study is not the first report on the molecular characterization of BKV in Sudan. However, most of the previous works have focused solely on the isolates associated with BKV nephropathy. Therefore, our study is the first of its kind to address the molecular characterization of BKV for PCa patients. Historically, BKV subtyping and subgrouping were based mainly on the phylogenetic analysis of some partial gene sequences (vp1 gene). While phylogenetic trees based on LTA sequences have been recently introduced and validated [20Luo C, Bueno M, Kant J, Martinson J, Randhawa P. Genotyping schemes for polyomavirus BK, using gene-specific phylogenetic trees and single nucleotide polymorphism analysis. J Virol 2009; 83(5): 2285-97.
[http://dx.doi.org/10.1128/JVI.02180-08] [PMID: 19109389] ]. This study was based only on the large T antigen gene product 176 bp to construct the phylogenetic tree. All the sequences of the samples were analyzed using various bioinformatics tools in order to understand genotype variability, molecular epidemiology and evolutionary adaptability of circulating strains. The purpose for focusing on the LTAg gene in our study was to determine the correlation between the BKV and prostate cancer in our previous work and to find a positive correlation between the presence of the virus and PCa and it is well known that LTAg promotes cellular transformation by interfering with the tumor suppressor functions of p53 [4Delbue S, Matei DV, Carloni C, et al. Evidence supporting the association of polyomavirus BK genome with prostate cancer. Med Microbiol Immunol (Berl) 2013; 202(6): 425-30.
[http://dx.doi.org/10.1007/s00430-013-0304-3] [PMID: 23821367] -6Provenzano M, Keller P. The potential therapeutic usefulness of targeting BK polyoma virus in prostate cancer. Chemo Open Access 2015; 5: 1.]. Thus, in this study, a point mutation was determined that could arise in this gene, resulting in antigen change. The latter event may affect the effectiveness of the antigen to bind with the antigen suppressing the tumor; this effect can be positive or negative.

In this study, there were two remarkable findings. Firstly, 3 out of 5 PCa BKV isolates were clustered into the same clade with Iranian and Japanese isolates. A previous study in Sudan revealed the BKV LTAg sequences among kidney transplant recipients and reported that three of these isolates had nucleotide similarities with the Japanese isolates. However, their study differed from our study in the clinical source of the isolates. They obtained their isolates from the urine samples of kidney transplant patients while our isolates were originated from the tissue samples of the patients with a prostate disorder. Moreover, in Africa, our isolates have similar nucleotides sequences with Zambian BKV isolate, while Ibrahim et al. found a nucleotide similarity between their isolate and the Ethiopian isolate [30Ibrahim AH, Hisham NA, Yousif FH, et al. Molecular characterization of polyomaviruses (BKV, JCV) in a symptomatic kidney transplant recipients in sudan. AJIDM 2016; 4: 44-51.]. Another significant point was that among our studied isolates, 5 BKV isolates (2 from PCa patients, 2 from BPH patients and 1 from the urine of BPH patients) were clustered together in the same clade with the USA strain, however, the similarity was 99%. Having 5 isolates in the same clade despite controlling the PCR condition and preventing DNA contamination can be considered as a strong evidence that the Sudanese isolates are likely to be closer to each other and could show genetic characteristics different from other strains found in the gene bank. Another point which may support our statement is the presence of point mutation which appears to be silent in some isolates, such as 2, 7 and 6 or transversion mutation like isolates 5 and 8. This latest finding may need further investigation considering the fact that both the mutant BKV isolates were from the hospitals in South and West Sudan which are closely related regions.

The histological identification Gleason scores for prostate cancer samples 1, 2, 3, 4 and 5 were 7, 6, 8, 7 and 8, respectively. Only isolate 5, which had a Gleason score of 8, showed a mismatched mutation, while other scores showed identical sequences upon alignment except for isolate 2 in which there was a silent mutation that had no significant impact on the translated amino acids. The association of the transversion mutation with the highest Gleason score in this study could raises a question whether the mutant variant of the BKV leads to a more aggressive type of PCa or not? In order to answer this question, further studies should be conducted by the inclusion of more BKV isolates from patients with prostate cancer grades.

Our result documented the circulation of one subtype (BKV Subtype 1) among Sudanese PCa and BPH patients with different age and geographical affiliations, regardless of the prostate Cancer Gleason scores. This finding was similar to that obtained by Maryam et al. in 2018 who concluded that all the BKV strains which were included in their study were classified under subtype 1. This is not surprising because most previous studies have revealed that despite different clinical samples, subtype I predominates in all the geographical regions of the world with higher frequencies, while subtype IV occurs at lower rates; and subtypes II and III occur rarely [31Chehadeh W, Kurien SS, Nampoory MR. Molecular characterization of BK and JC viruses circulating among potential kidney donors in kuwait. BioMed Res Int 2013; 2013683464
[http://dx.doi.org/10.1155/2013/683464] [PMID: 23936831] -36Ambalathingal GR, Francis RS, Smyth MJ, Smith C, Khanna R. BK polyomavirus: clinical aspects, immune regulation, and emerging therapies. Clin Microbiol Rev 2017; 30(2): 503-28.
[http://dx.doi.org/10.1128/CMR.00074-16] [PMID: 28298471] ]. Unfortunately, as only the LTAg gene was used to conduct the phylogenetic analysis, the BKV subtype 1 subgroups were not determined. The subgrouping of the BKV subtypes will be a very interesting topic for future studies.

In Sudan, there were no phylogenetic analysis reports on the BKV genome detected among patients with PCa and BPH tissues. In our study, more than 65% and 37% of studied samples were derived from PCa and BPH patients. Studies on the diversity of large T antigen gene sequences may have an implicit role in understanding the development of PCa by BKV because the LTAg gene, together with a small T antigen gene, produces antigens that are responsible for virus carcinogenesis [18Yu J, Boyapati A, Rundell K. Critical role for SV40 small-t antigen in human cell transformation. Virology 2001; 290(2): 192-8.
[http://dx.doi.org/10.1006/viro.2001.1204] [PMID: 11883184] , 19Maurizio P, Etienne XK. The potential therapeutic usefulness of targeting BK Polyomavirusin prostate cancer. Chemo Open Access 2015; 5: 1-2.]. Our study had some limitations. Due to a lack of funding, only LTAg gene sequences were used in this study. A few numbers of isolates were included in this study. Therefore, the results may not be generalized. Nevertheless, two new transversion mutations among all the studied patients were consistently reported. This latest finding has not been reported before among Sudanese patients, which increases the value of our findings.

The results of this study emphasized the importance of early detection and characterization of newly emerging genotypes. It was concluded that all the BKV isolates which circulate among Sudanese prostate tumoral patients belonged to subtype 1, andtwo isolates showed transversion mutations within the large T antigen amino acids sequence (positions 253), in addition to the presence of a silent mutation in some other isolates. These findings highlighted the need for the molecular detection and typing of BKV in Sudan for a better demonstration of the relationship between the virus and PCa. Moreover, our sequence analysis was done on the Large T antigen gene rather than the VP1 gene which is used as a standard target for the determination of BKV subtypes. Therefore, further studies must be conducted on molecular genotyping of BKV in Sudan by studying phylogeny through the amplification of both the above-mentioned genes with the inclusion of more BKV isolates,focusing on prostate cancer grades and patient age in addition to their geographical affiliations as well as the performance of a cell culture, transfection and BKV rescue to demonstrate that DNA BKV belonged to initially replicating BKV viruses. These may enable a better demonstration of the correlation between the BKV isolates and PCa.