Sir--Daniela Capello and colleagues (Jan 4, p 88)¹ used PCR to determine the presence of simian virus 40 (SV40) in archival lymphoma samples from patients in Italy and Spain. They report a detection rate of 3·4% using two different sets of primers against the N-terminus of the large tumour antigen (Tag) gene, but no sequences were detected with primers against the C-terminal region. On the basis that sequences needed to be amplified by all three primer sets for a sample to be regarded as positive, the authors concluded that "no case of lymphoproliferative disorders scored positive for SV40".

 

Different SV40 strains are known to exist in the human population.^2,3 These strains can be distinguished on the basis of nucleotide differences in the C-terminal variable domain of the Tag gene. Our experience is that primers directed at the Tag C-terminus seldom work as well as those at the N-terminus. DNA quality must be very good to produce the relatively large C-terminal amplicons. Also, these sequences could be less well conserved among the natural isolates found in tumours than in the isolates that were sequenced, resulting in poor primer binding in tumour samples.

 

Capello and colleagues also suggest that "geographic variability cannot be ascribed to differences in the contamination of poliovaccines, since contaminated lots were distributed in the USA and southern Europe over a similar period". In fact, the number of people infected with SV40 through the use of contaminated polio vaccines is not known.^4,5 In the USA, not all vaccine lots were contaminated with SV40, formalin inactivation was expected to reduce the titre of live SV40 in the lots that were contaminated, and successful infection rates by live SV40 are unknown.^4,5 One cannot assume that contaminated vaccines distributed in the USA and Europe were comparable with respect to SV40.

 

The Institute of Medicine of the US National Academies has concluded that "the biological evidence is strong that SV40 is a transforming virus" and that "the biological evidence is of moderate strength that SV40 exposure could lead to cancer in humans under natural conditions including lymphomas".⁴ Further, the Institute of Medicine recommended "targeted biological research", including "further study of the transmissibility of SV40 in humans"⁴ to fill gaps in our knowledge of the pathogenesis of SV40 in people today. RAV received the 2001 Junior Faculty Development Award from GlaxoSmithKline, and the 2002 Translational Research Award from the Leukemia and Lymphoma Society.

*Regis A Vilchez, Janet S Butel

 

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Departments of *Medicine (RAV) and Molecular Virology and Microbiology (RAV, JSB), Section of Infectious Diseases, Baylor College of Medicine, Houston, TX 77030, USA (e-mail:rvilchez@bcm.tmc.edu)

 

 

1 Capello D, Rossi D, Gaudino G, Carbone A, Gaidano G. Simian virus 40 infection in lymphoproliferative disorders.  Lancet 2003; 361: 88-89. [Text]

 

 

2 Stewart AR, Lednicky JA, Butel JS. Sequence analyses of human tumor-associated SV40 DNAs and SV40 viral isolates from monkeys and humans.  J Neurovirol 1998; 4: 182-93. [PubMed]

 

 

3 Stewart AR, Lednicky JA, Benzick US, Tevethia MJ, Butel JS. Identification of a variable region at the carboxy terminus of SV40 large T-antigen.  Virology 1996; 221: 355-61. [PubMed]

 

 

4 Stratton K, Almario DA, McCormick MC. SV40 contamination of polio vaccine and cancer. Immunization Safety Review Committee, Board of Health Promotion and Disease Prevention, Institute of Medicine of the National Academies. Washington, DC: National Academies Press, 2002.

 

 

5 Rollison DE, Shah KV. The epidemiology of SV40 infection due to contaminated polio vaccines: relation of the virus to human cancer. In: Kahalili K, Stoner GL, eds. Human polyomaviruses: molecular and clinical perspectives. New York: Wiley-Liss, 2001: 561-84.

 

Authors' reply

 

Sir--Regis Vilchez and Janet Butel argue that molecular heterogeneity at the C-terminus of the Tag gene could have prevented viral detection by our PCR strategy. We minimised the risk of primer mismatching at the C-terminus by doing several independent PCR assays with different primer sets that anneal to sequences conserved among viral strains. The selected primer sets were compared with more than 30 different viral sequences deposited in Gene bank and derived from clinical isolates, and virtually none of the reported nucleotide differences affected the sequences recognised by our primers. Moreover, all our PCR assays targeting the C-terminus of the Tag gene readily amplified SV40 DNA from mesothelioma cases known to harbour SV40 infection and derived from the same geographical region as the lymphoma samples, thus ruling out the suggestion by Vilchez and Butel that tumour-specific polymorphisms might have prevented SV40 detection.

 

Vilchez and Butel rightly raise the point that the quality of the genomic DNA must be very good to produce the relatively large amplicons (in the 400-500 bp range) expected when targeting the C-terminus of the Tag gene. To exclude false-negative results, all tumour samples included in our study had been previously tested for PCR suitability by amplification of an 748 bp fragment of the BCL6 gene and an 859 bp fragment of the PAX5 gene. In all cases a high-quality amplification was achieved. Therefore, we feel that poor quality of the DNA samples did not affect our results.

 

Vilchez and Butel also emphasise the uncertainty of the prevalence of SV40 infection in the human population and the possibility that rates of SV40 infection might be different in the USA and in Europe. We feel that this issue will benefit from accurate serological studies, and we agree that these investigations need to be encouraged on a large scale.

 

We deliberately chose a detection sensitivity of 10^-4 for our assay, and were thus able to rule out clonal infection of the tumour population by SV40. This process characterises infection by other viruses that exert a direct oncogenic role in lymphomagenesis--ie, Epstein-Barr virus, human herpesvirus 8 (HHV8), and human T-cell lymphotropic virus type I--as well as SV40-induced lymphomagenesis in animals.^1,2

 

On the basis of the long-standing controversy about HHV8 in multiple myeloma,³ we wish to stress the importance of interlaboratory standardisation of SV40 detection approaches to allow a proper comparison of the results obtained from different institutions. The authors were supported by grants from Istituto Superiore di Sanità, Programma Nazionale di Ricerca sull'AIDS--Progetto Patologia, Clinica e Terapia dell'AIDS, Rome, Italy; by Cofin--MIUR, Rome, Italy; and by CNR-MIUR Progetto Strategico Oncologia.

Daniela Capello, Davide Rossi, Giovanni Gaudino, Antonino Carbone, *Gianluca Gaidano

 

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*Haematology Unit (DC, DR, GG) and Laboratory of Molecular Biology (GG), Department of Medical Sciences and IRCAD, Amedeo Avogadro University of Eastern Piedmont, 28100 Novara, Italy; and Division of Pathology, IRCCS-Centro di Riferimento Oncologico, Aviano, Italy (AC) (e-mail:gaidano@med.unipmn.it)

 

 

1 Gaidano G, Dalla-Favera R. Pathobiology of non-Hodgkin lymphomas. In Hoffman R, Benz EJ Jr, Shattil SJ, et al, eds. Hematology: basic principles and practice. New York: Churchill Livingstone, 2000: 1213-29.

 

 

2 Butel JS, Lednicky JA. Cell and molecular biology of simian virus 40: implications for human infection and disease.  J Natl Cancer Inst 1999; 91: 119-34. [PubMed]

 

 

3 Masood R, Zheng T, Tupule A, et al. Kaposi's sarcoma-associated herpesvirus infection and multiple myeloma.  Science 1997; 278: 1969-72. [PubMed]