JC virus genotyping using formalin-fixed, paraffin-embedded renal tissues

Recently genotyping of JC virus (JCV) DNA in renal tissue was reported to be useful to identify the geographic origin of unidentified cadavers. In the above study, autopsied tissue samples without storage or stored in a frozen state were used. This study examined JCV DNA sequence modifications caused by formalin-fixation, in an attempt to elucidate whether formalin-fixed, paraffin-embedded tissue samples can also be used to determine the genotypes of JCV DNA in the kidney. In four cases, a 610 bp typing region of the JCV genome was PCR-amplified from renal tissues stored for 1 year in three different states: frozen at -80 degrees C [Amaker, B.H., Chandler, F.W., Huey, L.O., Colwell, R.M., 1997. Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue. J. Forensic Sci., 1157-1159], formalin-fixed, paraffin-embedded [Ault, G.S., Stoner, G.L., 1992. Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences. J. Gen. Virol. 73, 2669-2678], and soaked in 5% formalin [Baksh, F.K., Finkelstein, S.D., Swalskey, P.A., Stoner, G.L., Ryschkewitsch, C.F., Randhawa, P.R., 2001. Molecular genotyping of BK and JC virus in human polyomavirus-associated interstitial nephritis after renal transplantation. Am. J. Kidney Dis. 38 (2), 354-365]. The amplified fragments were cloned, and the resultant clones were sequenced. In frozen samples, single sequences ('original' sequences) were detected in all cases. In formalin-fixed, paraffin-embedded samples, not only the original sequences but also those with 1-6 base substitutions were detected. From formalin-soaked samples, the original sequences and those with 1-5 and 10-13 substitutions were detected. The genotyping of JCV DNA was not hampered by the presence of 1-6 substitutions, but a shift in JCV genotypes was observed in sequences with 10-13 substitutions. Thus, it was concluded that the genotypes of JCV DNA in the kidney can be determined only with specimens stored in a frozen state or formalin-fixed for a short time.