122s Biochemical Society Transactions ( I99 I ) I9 Detection of herpes simplex virus type 1 DNA sequences in normal and Alzheimer's disease brain using plymerase chain reaction.
b
p
1
2
3
4
5
6
7
8
9
1
0
1
1
*
JAMIESON , NORMAN*g. M A I " f + , JOHN * CRASKE , GORWN K. WILCCCK , and RLTH F. ITZHAKI GORDON+$.
Molecular Neurobiology Laboratory, Cepartment of @tawtry and Visual Sciences, UMIST, Manchester, M60 lQD, UK. 'Department of Pathology, University of Bristol, Bristol, BS8 lTD, UK. "Department of yirology, Withington Hospital, Manchester, M20 8LK, LK. Department of Cdre of the Elderly, Frenchdy Hospitdl Bristol, BS16 lLE, UK. The aetiology and pathogenesis of AlLheimer's disease (AD)are unknown, in particuldr, whether or not environmental agents such as viruses play a role. However no convincing evidence has yet been found t o implicate an infectious pathogen in AD; wnether thi5 reflects the absence of such an agent or whether the Lick of sensitive techniques has precluded its detection, is uncertain. Here we are investigating a pssible corrcldtion ktween the presence of herpes siqlex virus type 1 (HbV-1) DNA sequences in human brain and the incidence of AD, using the method of polymerase chain redction (PCR). The predilection of HSV for those reyions of the brain which are the most severely affected in AD has led to spsculdtion that this virus may k implicated in AD [l] . HSV-1 has been considered d likely agent also because of its propensity to produce latent infections in neuronal cells and because of its ubiquity. PCR meets the criteria of both specificity and sensitivity required for the detpction of latent HSV and it represents a very great improvenetit over traditional methods of nucleic acid detection. We looked for the presence of a 110 base-pair (bp) fragment of the viral thymidine kinase (TK)ycne. As an internal control, to ensure that the DNA sample does not contain an inhibitor of PCR that could lead to artekdctual negative results a 267bp fragment ot a c c , l l u l a r gene hypoxanthine phosphoribosyl transterase (HPRT)was amplified simultaneously. Nucleic acids were separated by isopycnic centrifugation in caesium trifluoroacetate and DNA was purified by protedse digestion, phenol-chloroform extraction and ethanol precipitation. PCR was used to screen DNA from 17 autopsy brain specmns (derived frcm 8 AD patients and 5 normals) and in all cases both TK and HPRT sequences were detectahle. Cross-contamination was excluded since Vero negativcs and also a 'buffer blank' were TK-negative. Fig 1 shows a representative set of 7 specimens. DNA samples frcm HSV-infected and uninfected Vero cells were examined concurrently to provide HSV-postive and HSV-negative controls. In contrast to these results on brain DNA, in preliminary studies of DNA extracted from both AD and normal lymphocytes we have not found HSV sequences; therefore our results on brain cannot be an artefact due to virus in lymphocytes within blood vessels at the time of death. Our results, though unexpected, are not entirely surprising, as serological evidence has revealed that more than 90% of adults over the age of 60 have been infected with HSV [2] . Previous Southern blot, dot blot or in situ hybridisation studies have yielded conflicting results regarding the presence of E b i DNA in AD brain. In contrast, the very sensitive assay used here for detecting HSV DNA will provide an opportunity to
Fig. 1. Uctection of IISV DNA s(qucm.w i n liiuniiri brain spccimcns by plymcrasc, chdin rr~wctioii, followed by ayarose gel elect rophorcsis. Noirnal rnaic NM, female NF'; AL) nialc, ADM, female, RDF. Lane 1, flae 111 - diyc>sted x 174 DNA inilrkt-r; 1,anes2 and 3, AIIF, 91, niiu-frontal
b
p
1
2
3
4
5
6
7
8
9
1
0
1
1
*
JAMIESON , NORMAN*g. M A I " f + , JOHN * CRASKE , GORWN K. WILCCCK , and RLTH F. ITZHAKI GORDON+$.
Molecular Neurobiology Laboratory, Cepartment of @tawtry and Visual Sciences, UMIST, Manchester, M60 lQD, UK. 'Department of Pathology, University of Bristol, Bristol, BS8 lTD, UK. "Department of yirology, Withington Hospital, Manchester, M20 8LK, LK. Department of Cdre of the Elderly, Frenchdy Hospitdl Bristol, BS16 lLE, UK. The aetiology and pathogenesis of AlLheimer's disease (AD)are unknown, in particuldr, whether or not environmental agents such as viruses play a role. However no convincing evidence has yet been found t o implicate an infectious pathogen in AD; wnether thi5 reflects the absence of such an agent or whether the Lick of sensitive techniques has precluded its detection, is uncertain. Here we are investigating a pssible corrcldtion ktween the presence of herpes siqlex virus type 1 (HbV-1) DNA sequences in human brain and the incidence of AD, using the method of polymerase chain redction (PCR). The predilection of HSV for those reyions of the brain which are the most severely affected in AD has led to spsculdtion that this virus may k implicated in AD [l] . HSV-1 has been considered d likely agent also because of its propensity to produce latent infections in neuronal cells and because of its ubiquity. PCR meets the criteria of both specificity and sensitivity required for the detpction of latent HSV and it represents a very great improvenetit over traditional methods of nucleic acid detection. We looked for the presence of a 110 base-pair (bp) fragment of the viral thymidine kinase (TK)ycne. As an internal control, to ensure that the DNA sample does not contain an inhibitor of PCR that could lead to artekdctual negative results a 267bp fragment ot a c c , l l u l a r gene hypoxanthine phosphoribosyl transterase (HPRT)was amplified simultaneously. Nucleic acids were separated by isopycnic centrifugation in caesium trifluoroacetate and DNA was purified by protedse digestion, phenol-chloroform extraction and ethanol precipitation. PCR was used to screen DNA from 17 autopsy brain specmns (derived frcm 8 AD patients and 5 normals) and in all cases both TK and HPRT sequences were detectahle. Cross-contamination was excluded since Vero negativcs and also a 'buffer blank' were TK-negative. Fig 1 shows a representative set of 7 specimens. DNA samples frcm HSV-infected and uninfected Vero cells were examined concurrently to provide HSV-postive and HSV-negative controls. In contrast to these results on brain DNA, in preliminary studies of DNA extracted from both AD and normal lymphocytes we have not found HSV sequences; therefore our results on brain cannot be an artefact due to virus in lymphocytes within blood vessels at the time of death. Our results, though unexpected, are not entirely surprising, as serological evidence has revealed that more than 90% of adults over the age of 60 have been infected with HSV [2] . Previous Southern blot, dot blot or in situ hybridisation studies have yielded conflicting results regarding the presence of E b i DNA in AD brain. In contrast, the very sensitive assay used here for detecting HSV DNA will provide an opportunity to
Fig. 1. Uctection of IISV DNA s(qucm.w i n liiuniiri brain spccimcns by plymcrasc, chdin rr~wctioii, followed by ayarose gel elect rophorcsis. Noirnal rnaic NM, female NF'; AL) nialc, ADM, female, RDF. Lane 1, flae 111 - diyc>sted x 174 DNA inilrkt-r; 1,anes2 and 3, AIIF, 91, niiu-frontal
