Molecular and Cellular Probes (1992) 6, 23 1 -235
Effects of post-mortem autolysis on the detection of rabies virus genomic RNA and mRNA in mouse brain by using in
situ hybridization Alan C . Jackson* and Natalie E . Rintoul Departments of Medicine and Microbiology & Immunology, Queen's University, 78 Barrie St., Kingston, Ontario, Canada K7L 3J7 (Received 30 October 1991, Accepted 5 December 1991)
The effects of post-mortem autolysis were studied on the detection of rabies virus RNA in the brains of mice with experimental rabies by using in situ hybridization (ISH) . The brains of CVS-infected mice were subjected to autolytic periods in situ of up to 72 h . ISH was performed with 3H-labelled RNA probes for rabies virus glycoprotein gene genomic RNA and mRNA . During the post-mortem period there was progressive loss of signals for genomic RNA and mRNA, which was greater for mRNA . ISH signals in perikarya also changed for genomic RNA from a multifocal to a diffuse distribution during the post-mortem period . Rabies virus antigen was better preserved during the autolytic period . Effects of the agonal state, degradation of RNA by ribonucleases, and diffusion of RNA out of cells prior to fixation could explain the loss of ISH signals in post-mortem tissues . KEYWORDS :
autolysis, nucleic acid hybridization, post-mortem change, rabies .
INTRODUCTION
MATERIALS AND METHODS
Viral nucleic acids and cellular mRNAs can be locaVirus lized in tissues with in situ hybridization (ISH) .''2 ISH is frequently performed on post-mortem human tissues The CVS-11 strain of fixed rabies virus was obtained in both virological and gene expression studies . Alfrom Dr William H . Wunner (Wistar Institute, Philaterations of cellular components, including nucleic delphia, Pennsylvania) . Stock CVS was grown in BIHKacids and proteins, occur during the post-mortem 21 cells to a titre of 4 . 2 x 10' plaque-forming units autolytic period . The effects of post-mortem autolysis (pfu) ml - '. on ISH signals have not been examined experimentally except in a single study on a cellular mRNA by Arai et a! . 3 In the present study, the effects of postAnimals and inoculations mortem autolysis on the detection of rabies virus genomic RNA and mRNA were studied in the brains Six-week-old female ICR mice (Charles River Canada, of mice with experimental rabies, and they were Inc., St-Constant, Quebec) were used . Mice were compared with the effects on the detection of rabies inoculated intracerebrally with 9 . 3 x 105 pfu of CVS in virus antigen with immunoperoxidase staining . 0. 03 ml of phosphate-buffered saline (PBS) with 2% * Author to whom correspondence should be addressed .
0890-8508/92/030231 +05 $03 .00/0
231
© 1992 Academic Press Limited
232
A. C . Jackson and N. E . Rintoul
fetal bovine serum (FBS) . Control mice were inoculated with 0 . 03 ml of PBS with 2% FBS .
RESULTS ISH signals for rabies virus genomic RNA and mRNA
Preparation of tissue sections
All mice were sacrificed
4 days after inoculation, which was prior to the development of neurological signs of rabies . Three CVS-infected mice and a control mouse were anaesthetized with methoxyflurane and perfused with buffered 4% paraformaldehyde on day 4 . The brains were removed and immersion-fixed
in the same fixative for
18 h at 4© C . The remaining mice were sacrificed by inhalation of a fatal dose of methoxyflurane . The brains were left in situ during the post-mortem interval at room temperature (27 © C) . The brains of four infected mice and a control mouse were removed immediately and at 12, 24, 48, and 72 h after sacrifice . The brains were immersion-fixed in buffered 4% paraformaldehyde for 18 h at 4©C, dehydrated, and embedded in paraffin . Coronal sections (6 °m) of brain were cut at multiple levels on a microtome .
Immunoperoxidase staining Tissue sections were stained for rabies virus antigen by the avidin-biotin-peroxidase method as previously described by Jackson and Wunner 4
ISH signal intensities for rabies virus genomic RNA and mRNA were similar in mice perfused with fixative and in unperfused mice without a period of postmortem autolysis . As previously reported," probes for genomic RNA showed a multifocal distribution of autoradiographic grains over the perikarya and dendrites of many infected neurons, while probes for mRNA showed a diffuse distribution of grains over the perikarya and proximal dendrites (Figs la and b) . More signal was detected with probes for mRNA than genomic RNA (Figs la and b) . After 12 h of post-mortem autolysis there was a mild reduction in signals for mRNA, but no reduction for genomic RNA (data not shown) . Genomic RNA had a less prominent multifocal distribution, while the distribution of mRNA remained diffuse (data not shown) . By 24 h there was a further reduction in signals for mRNA, and genomic RNA was not reduced (Figs 1c and d) . The distribution of signal for genomic RNA became diffuse after 24 h and it became indistinguishable from that of mRNA . By 48 h there was a marked reduction in ISH signals for mRNA and a moderate reduction for genomic RNA (Figs le and f) . There was very little signal for either genomic RNA or 72 h, although signals were greater genomic RNA than mRNA (data not shown) .
mRNA at
for
In situ hybridization Rabies virus antigen ISH hybridization was performed as previously de-
scribed by`jackson and Wunner 4 with minor modifications . A cDNA clone containing the coding sequence for the rabies virus glycoprotein (G) of the ERA strain of , fixed rabies virus (obtained from Connaught Research Institute, Willowdale, Ontario)' was
used to prepare radiolabelled RNA probes . The 3 Hlabelled
probes
had
specific
activities
of
6 .0 x 10 7 dpm °g - ' . An irrelevant control template (Riboprobe Gemini positive control template, Pro-
There was progressive loss in the amount of rabies virus antigen over the 72 h autolytic period . There was a moderate reduction in the amount of immunoperoxidase staining in brains subjected to 72 h of post-mortem autolysis compared with brains fixed immediately after death (Figs 1g and h) . This reduction was much less than for ISH signals for genomic RNA and mRNA . Antigen remained localized in the perikarya and dendrites of infected neurons .
mega) was used to prepare 3 H-labelled RNA transcripts as a control of the specificity of the hybridization . The slide pretreatment, hybridization, posthybridization washes, and autoradiography were performed as previously described .' The autoradiographic exposure was for 6 days .
DISCUSSION Post-mortem human and animal tissues are frequently examined for the presence of specific nucleic acids and proteins (antigens) . During the post-mortem interval there is autolysis with alterations in cellular components that could impair the detection of nucleic acids and antigens in tissues . The present study has shown that in situ hybridization signals for
Effects of post-mortem autolysis on rabies virus RNA
233
Fig . 1 . In situ hybridization without a period of post-mortem autolysis for rabies virus genomic RNA (a) and mRNA (b) in mouse Purkinje cells . Grains had a multifocal distribution in perikarya for genomic RNA and a diffuse distribution for mRNA, and signals were greater for mRNA . After 24 h of post-mortem autolysis ISH signals for genomic RNA (c) were more diffuse in distribution than at earlier time points (a), and signals for mRNA (d) were reduced . After 48 h of post-mortem autolysis there was reduction in the ISH signals for both genomic RNA (e) and mRNA (f), which was more marked for mRNA . Rabies virus antigen in Purkinje cells fixed immediately after death (g) and after a post-mortem interval of 72 h (h) . There was moderate reduction in the amount of antigen over this period . Hematoxylin (a-f) and immunoperoxidase-haematoxylin (g and h) X 1200) . rabies virus RNA in mouse brain are more sensitive to post-mortem autolysis than signals for rabies virus antigen detected with the immunoperoxidase technique. Pelstring et al.' recently reported remarkable preservation of most antigens during autolysis, although they found differential preservation of various cellular markers . Losses of ISH signals were mild in this study over 12-24 h post-mortem, but they became marked after 48-72 h . The human body is
usually refrigerated (2-4° C) within about 4 h after death until the tissues are dissected, and there is typically a post-mortem delay of 12-18 h . 7 Hence, a major loss of ISH signals would not be expected after a post-mortem period of less than 24 h in humans if changes are similar in humans as in this model . However, the human brain cools more slowly than the mouse brain' and it takes longer for fixative to penetrate and fix the relatively large human brain .
234
A. C . Jackson and N . E. Rintoul
ISH signals for rabies virus genomic RNA were found to be more resistant to post-mortem autolysis than rabies virus mRNA . There is a relatively close association of proteins with genomic RNA in the ribonucleoprotein complex 9' 10 compared to mRNA . The proteins likely provide greater resistance for genomic RNA against degradation by ribonucleases than mRNA . Because of the larger size of genomic RNA and its tendency for aggregation, it may also diffuse out of infected cells , prior to fixation less readily than mRNA. A large number of studies have examined the effects of the post-mortem period on RNA in tissues . In general, RNA is quite stable for a prolonged period after death . There is a mild or no reduction in total cellular RNA in humans and animals after postmortem intervals of up to 134 h .1-1S The biological activity of mRNAs has been assessed with in vitro translation systems,", ", " including two-dimensional gel analysis of the proteins synthesized in vitro . These studies show little degradation of individual mRNA species after post-mortem periods of up to 84 h in both human and rat brains . Ermine et al." extracted rabies virus RNA from CVS-infected mouse brains and performed Northern blot analyses with cDNA probes, and they did not find significant degradation of rabies virus RNA until at least 120 h post-mortem . Arai et al.' assessed the effects of a post-mortem interval at room temperature on ISH signals for vasopressin mRNA in rat brains. They found reduced signals after 8 h, and signals became markedly reduced after 24 h . Northern blot analyses also showed a marked reduction in the amount of mRNA between 8 and 24 h post-mortem . It is uncertain whether vasopressin mRNA is particularly sensitive to degradation by ribonucleases, or whether a difference in methodologies accounts for the variability in the resistance of specific mRNA species to degradation . These studies show there is reduction of in situ hybridization signals during a 72 h period of postmortem autolysis . If the post-mortem effects on rabies virus RNA are similar in human brains to this experimental murine model, then they do not adequately explain the low signals observed for rabies virus mRNA in brains from cases of human rabies .' Effects of the agonal state in rabies, 2 '17'78 degradation of RNA by ribonucleases, and diffusion of RNA out of cells prior to fixation could explain the loss of ISH signals in post-mortem tissues .
ACKNOWLEDGEMENTS We thank Connaught Research Institute for the cDNA
clone containing the coding sequence for the rabies virus glycoprotein and K . M. Charlton (Animal Diseases Research Institute, Nepean, Ontario) for the anti-rabies virus serum . The secretarial assistance of Martha Steacy is gratefully acknowledged . This work was supported by grant MA-10068 from the Medical Research Council of Canada and the Violet E . Powell Fund (Queen's University) .
REFERENCES 1 . Haase, A . T. (1987). Analysis of viral infections by in situ hybridization . In In Situ Hybridization: Applications to Neurobiology . (Valentino, K . L ., Eberwine, J . H . & Barchas, J . D ., eds .) pp. 197-219. New York : Oxford University Press . 2 . Harrison, P. J . & Pearson, R. C . A . (1990). In situ hybridization histochemistry and the study of gene expression in the human brain . Progress in Neurobiology 34, 271312 . 3 . Arai, H ., Noguchi, I ., Sagi, N ., Moroji, T . & lizuka, R . (1989) . A study of non-isotopic in situ hybridization histochemistry on postmortem changes in vasopressin mRNA in rat brain . Neuroscience Letters 103, 127-32 . 4. Jackson, A . C . & Wunner, W . H. (1991) . Detection of rabies virus genomic RNA and mRNA in mouse and human brains by using in situ hybridization . Journal of Virology 65, 2839-44. 5 . Malek, L . T., Soostmeyer, G ., Garvin, R . T . & James, E . (1984). The rabies glycoprotein gene is expressed in Escherichia coli as a denatured polypeptide . In Modern Approaches to Vaccines : Molecular and Chemical Basis of Virus Virulence and Immunogenicity . (Chanock, R . M . & Lerner, R . A ., eds .) pp . 203-208 . Cold Spring Harbor, New York : Cold Spring Harbor Laboratory . 6 . Pelstring, R . J ., Alfred, D . C., Esther, R . J ., Lampkin, S . R . & Banks, P . M . (1991) . Differential antigen preservation during tissue autolysis . Human Pathology 22, 237-41 . 7 . Whitehouse, P. J., Lynch, D . & Kuhar, M . J . (1984) . Effects of postmortem delay and temperature on neurotransmitter receptor binding in a rat model of the human autopsy process . Journal of Neurochemistry 43, 553-9 . 8 . Spokes, E . G . S . & Koch, D . J . (1978) . Post-mortem stability of dopamine, glutamate decarboxylase and choline acetyltransferase in the mouse brain under conditions simulating the handling of human autopsy material . Journal of Neurochemistry 31, 381-3 . 9 . Wunner, W . H ., Larson, J . K ., Dietzschold, B . & Smith, C . L. (1988). The molecular biology of rabies viruses . Reviews of Infectious Diseases 10 (Suppl 4), 5771-5784 . 10 . Wunner, W . H . (1991) . The chemical composition and molecular structure of rabies viruses . In The Natural History of Rabies . ( Baer, G . M ., ed .) 2nd edn, pp . 31-67 . Boca Raton, Florida : CRC Press . 11 . Taylor, C . R., Carter, G . I ., Crow, T. J . et al. (1986) . Recovery and measurement of specific RNA species from postmortem brain tissue : A general reduction in Alzheimer's disease detected by molecular hybridization . Experimental and Molecular Pathology 44, 111-6 . 12 . Morrison, M . R . & Griffin, W . S .T. (1981) . The isolation and in vitro translation of undegraded messenger RNAs
Effects of post-mortem autolysis on rabies virus RNA
from
human postmortem
brain .
Analytical
Bio-
chemistry 113, 318-24.
13 . Guillemette, J . G., Wong, L ., McLachlan, D . R . C . & Lewis, P . N . (1986) . Characterization of messenger RNA from the cerebral cortex of control and Alzheimerafflicted brain . Journal of Neurochemistry 47, 987-97 . 14 . Perrett, C . W., Marchbanks, R . M. & Whatley, S . A . (1988) . Characterisation of messenger RNA extracted post-mortem from the brains of schizophrenic, depressed and control subjects . Journal of Neurology, Neurosurgery, and Psychiatry 51, 325-31 . 15 . Johnson, S . A ., Morgan, D . G . & Finch, C . E . (1986) .
235
Extensive postmortem stability of RNA from rat and human brain . Journal of Neuroscience Research 16, 267-80. 16 . Ermine, A ., Tordo, N . & Tsiang, H . (1988). Rapid diagnosis of rabies infection by means of a dot hybridization assay . Molecular and Cellular Probes 2, 75-82 . 17 . Hattwick, M . A . W . (1974). Human rabies . Public Health Review 3, 229-74 . 18 . Perry, E . K ., Perry, R . H . & Tomlinson, B . E . (1982) . The influence of agonal status on some neurochemical activities of postmortem human brain tissue. Neuroscience Letters 29, 303-7.
Effects of post-mortem autolysis on the detection of rabies virus genomic RNA and mRNA in mouse brain by using in
situ hybridization Alan C . Jackson* and Natalie E . Rintoul Departments of Medicine and Microbiology & Immunology, Queen's University, 78 Barrie St., Kingston, Ontario, Canada K7L 3J7 (Received 30 October 1991, Accepted 5 December 1991)
The effects of post-mortem autolysis were studied on the detection of rabies virus RNA in the brains of mice with experimental rabies by using in situ hybridization (ISH) . The brains of CVS-infected mice were subjected to autolytic periods in situ of up to 72 h . ISH was performed with 3H-labelled RNA probes for rabies virus glycoprotein gene genomic RNA and mRNA . During the post-mortem period there was progressive loss of signals for genomic RNA and mRNA, which was greater for mRNA . ISH signals in perikarya also changed for genomic RNA from a multifocal to a diffuse distribution during the post-mortem period . Rabies virus antigen was better preserved during the autolytic period . Effects of the agonal state, degradation of RNA by ribonucleases, and diffusion of RNA out of cells prior to fixation could explain the loss of ISH signals in post-mortem tissues . KEYWORDS :
autolysis, nucleic acid hybridization, post-mortem change, rabies .
INTRODUCTION
MATERIALS AND METHODS
Viral nucleic acids and cellular mRNAs can be locaVirus lized in tissues with in situ hybridization (ISH) .''2 ISH is frequently performed on post-mortem human tissues The CVS-11 strain of fixed rabies virus was obtained in both virological and gene expression studies . Alfrom Dr William H . Wunner (Wistar Institute, Philaterations of cellular components, including nucleic delphia, Pennsylvania) . Stock CVS was grown in BIHKacids and proteins, occur during the post-mortem 21 cells to a titre of 4 . 2 x 10' plaque-forming units autolytic period . The effects of post-mortem autolysis (pfu) ml - '. on ISH signals have not been examined experimentally except in a single study on a cellular mRNA by Arai et a! . 3 In the present study, the effects of postAnimals and inoculations mortem autolysis on the detection of rabies virus genomic RNA and mRNA were studied in the brains Six-week-old female ICR mice (Charles River Canada, of mice with experimental rabies, and they were Inc., St-Constant, Quebec) were used . Mice were compared with the effects on the detection of rabies inoculated intracerebrally with 9 . 3 x 105 pfu of CVS in virus antigen with immunoperoxidase staining . 0. 03 ml of phosphate-buffered saline (PBS) with 2% * Author to whom correspondence should be addressed .
0890-8508/92/030231 +05 $03 .00/0
231
© 1992 Academic Press Limited
232
A. C . Jackson and N. E . Rintoul
fetal bovine serum (FBS) . Control mice were inoculated with 0 . 03 ml of PBS with 2% FBS .
RESULTS ISH signals for rabies virus genomic RNA and mRNA
Preparation of tissue sections
All mice were sacrificed
4 days after inoculation, which was prior to the development of neurological signs of rabies . Three CVS-infected mice and a control mouse were anaesthetized with methoxyflurane and perfused with buffered 4% paraformaldehyde on day 4 . The brains were removed and immersion-fixed
in the same fixative for
18 h at 4© C . The remaining mice were sacrificed by inhalation of a fatal dose of methoxyflurane . The brains were left in situ during the post-mortem interval at room temperature (27 © C) . The brains of four infected mice and a control mouse were removed immediately and at 12, 24, 48, and 72 h after sacrifice . The brains were immersion-fixed in buffered 4% paraformaldehyde for 18 h at 4©C, dehydrated, and embedded in paraffin . Coronal sections (6 °m) of brain were cut at multiple levels on a microtome .
Immunoperoxidase staining Tissue sections were stained for rabies virus antigen by the avidin-biotin-peroxidase method as previously described by Jackson and Wunner 4
ISH signal intensities for rabies virus genomic RNA and mRNA were similar in mice perfused with fixative and in unperfused mice without a period of postmortem autolysis . As previously reported," probes for genomic RNA showed a multifocal distribution of autoradiographic grains over the perikarya and dendrites of many infected neurons, while probes for mRNA showed a diffuse distribution of grains over the perikarya and proximal dendrites (Figs la and b) . More signal was detected with probes for mRNA than genomic RNA (Figs la and b) . After 12 h of post-mortem autolysis there was a mild reduction in signals for mRNA, but no reduction for genomic RNA (data not shown) . Genomic RNA had a less prominent multifocal distribution, while the distribution of mRNA remained diffuse (data not shown) . By 24 h there was a further reduction in signals for mRNA, and genomic RNA was not reduced (Figs 1c and d) . The distribution of signal for genomic RNA became diffuse after 24 h and it became indistinguishable from that of mRNA . By 48 h there was a marked reduction in ISH signals for mRNA and a moderate reduction for genomic RNA (Figs le and f) . There was very little signal for either genomic RNA or 72 h, although signals were greater genomic RNA than mRNA (data not shown) .
mRNA at
for
In situ hybridization Rabies virus antigen ISH hybridization was performed as previously de-
scribed by`jackson and Wunner 4 with minor modifications . A cDNA clone containing the coding sequence for the rabies virus glycoprotein (G) of the ERA strain of , fixed rabies virus (obtained from Connaught Research Institute, Willowdale, Ontario)' was
used to prepare radiolabelled RNA probes . The 3 Hlabelled
probes
had
specific
activities
of
6 .0 x 10 7 dpm °g - ' . An irrelevant control template (Riboprobe Gemini positive control template, Pro-
There was progressive loss in the amount of rabies virus antigen over the 72 h autolytic period . There was a moderate reduction in the amount of immunoperoxidase staining in brains subjected to 72 h of post-mortem autolysis compared with brains fixed immediately after death (Figs 1g and h) . This reduction was much less than for ISH signals for genomic RNA and mRNA . Antigen remained localized in the perikarya and dendrites of infected neurons .
mega) was used to prepare 3 H-labelled RNA transcripts as a control of the specificity of the hybridization . The slide pretreatment, hybridization, posthybridization washes, and autoradiography were performed as previously described .' The autoradiographic exposure was for 6 days .
DISCUSSION Post-mortem human and animal tissues are frequently examined for the presence of specific nucleic acids and proteins (antigens) . During the post-mortem interval there is autolysis with alterations in cellular components that could impair the detection of nucleic acids and antigens in tissues . The present study has shown that in situ hybridization signals for
Effects of post-mortem autolysis on rabies virus RNA
233
Fig . 1 . In situ hybridization without a period of post-mortem autolysis for rabies virus genomic RNA (a) and mRNA (b) in mouse Purkinje cells . Grains had a multifocal distribution in perikarya for genomic RNA and a diffuse distribution for mRNA, and signals were greater for mRNA . After 24 h of post-mortem autolysis ISH signals for genomic RNA (c) were more diffuse in distribution than at earlier time points (a), and signals for mRNA (d) were reduced . After 48 h of post-mortem autolysis there was reduction in the ISH signals for both genomic RNA (e) and mRNA (f), which was more marked for mRNA . Rabies virus antigen in Purkinje cells fixed immediately after death (g) and after a post-mortem interval of 72 h (h) . There was moderate reduction in the amount of antigen over this period . Hematoxylin (a-f) and immunoperoxidase-haematoxylin (g and h) X 1200) . rabies virus RNA in mouse brain are more sensitive to post-mortem autolysis than signals for rabies virus antigen detected with the immunoperoxidase technique. Pelstring et al.' recently reported remarkable preservation of most antigens during autolysis, although they found differential preservation of various cellular markers . Losses of ISH signals were mild in this study over 12-24 h post-mortem, but they became marked after 48-72 h . The human body is
usually refrigerated (2-4° C) within about 4 h after death until the tissues are dissected, and there is typically a post-mortem delay of 12-18 h . 7 Hence, a major loss of ISH signals would not be expected after a post-mortem period of less than 24 h in humans if changes are similar in humans as in this model . However, the human brain cools more slowly than the mouse brain' and it takes longer for fixative to penetrate and fix the relatively large human brain .
234
A. C . Jackson and N . E. Rintoul
ISH signals for rabies virus genomic RNA were found to be more resistant to post-mortem autolysis than rabies virus mRNA . There is a relatively close association of proteins with genomic RNA in the ribonucleoprotein complex 9' 10 compared to mRNA . The proteins likely provide greater resistance for genomic RNA against degradation by ribonucleases than mRNA . Because of the larger size of genomic RNA and its tendency for aggregation, it may also diffuse out of infected cells , prior to fixation less readily than mRNA. A large number of studies have examined the effects of the post-mortem period on RNA in tissues . In general, RNA is quite stable for a prolonged period after death . There is a mild or no reduction in total cellular RNA in humans and animals after postmortem intervals of up to 134 h .1-1S The biological activity of mRNAs has been assessed with in vitro translation systems,", ", " including two-dimensional gel analysis of the proteins synthesized in vitro . These studies show little degradation of individual mRNA species after post-mortem periods of up to 84 h in both human and rat brains . Ermine et al." extracted rabies virus RNA from CVS-infected mouse brains and performed Northern blot analyses with cDNA probes, and they did not find significant degradation of rabies virus RNA until at least 120 h post-mortem . Arai et al.' assessed the effects of a post-mortem interval at room temperature on ISH signals for vasopressin mRNA in rat brains. They found reduced signals after 8 h, and signals became markedly reduced after 24 h . Northern blot analyses also showed a marked reduction in the amount of mRNA between 8 and 24 h post-mortem . It is uncertain whether vasopressin mRNA is particularly sensitive to degradation by ribonucleases, or whether a difference in methodologies accounts for the variability in the resistance of specific mRNA species to degradation . These studies show there is reduction of in situ hybridization signals during a 72 h period of postmortem autolysis . If the post-mortem effects on rabies virus RNA are similar in human brains to this experimental murine model, then they do not adequately explain the low signals observed for rabies virus mRNA in brains from cases of human rabies .' Effects of the agonal state in rabies, 2 '17'78 degradation of RNA by ribonucleases, and diffusion of RNA out of cells prior to fixation could explain the loss of ISH signals in post-mortem tissues .
ACKNOWLEDGEMENTS We thank Connaught Research Institute for the cDNA
clone containing the coding sequence for the rabies virus glycoprotein and K . M. Charlton (Animal Diseases Research Institute, Nepean, Ontario) for the anti-rabies virus serum . The secretarial assistance of Martha Steacy is gratefully acknowledged . This work was supported by grant MA-10068 from the Medical Research Council of Canada and the Violet E . Powell Fund (Queen's University) .
REFERENCES 1 . Haase, A . T. (1987). Analysis of viral infections by in situ hybridization . In In Situ Hybridization: Applications to Neurobiology . (Valentino, K . L ., Eberwine, J . H . & Barchas, J . D ., eds .) pp. 197-219. New York : Oxford University Press . 2 . Harrison, P. J . & Pearson, R. C . A . (1990). In situ hybridization histochemistry and the study of gene expression in the human brain . Progress in Neurobiology 34, 271312 . 3 . Arai, H ., Noguchi, I ., Sagi, N ., Moroji, T . & lizuka, R . (1989) . A study of non-isotopic in situ hybridization histochemistry on postmortem changes in vasopressin mRNA in rat brain . Neuroscience Letters 103, 127-32 . 4. Jackson, A . C . & Wunner, W . H. (1991) . Detection of rabies virus genomic RNA and mRNA in mouse and human brains by using in situ hybridization . Journal of Virology 65, 2839-44. 5 . Malek, L . T., Soostmeyer, G ., Garvin, R . T . & James, E . (1984). The rabies glycoprotein gene is expressed in Escherichia coli as a denatured polypeptide . In Modern Approaches to Vaccines : Molecular and Chemical Basis of Virus Virulence and Immunogenicity . (Chanock, R . M . & Lerner, R . A ., eds .) pp . 203-208 . Cold Spring Harbor, New York : Cold Spring Harbor Laboratory . 6 . Pelstring, R . J ., Alfred, D . C., Esther, R . J ., Lampkin, S . R . & Banks, P . M . (1991) . Differential antigen preservation during tissue autolysis . Human Pathology 22, 237-41 . 7 . Whitehouse, P. J., Lynch, D . & Kuhar, M . J . (1984) . Effects of postmortem delay and temperature on neurotransmitter receptor binding in a rat model of the human autopsy process . Journal of Neurochemistry 43, 553-9 . 8 . Spokes, E . G . S . & Koch, D . J . (1978) . Post-mortem stability of dopamine, glutamate decarboxylase and choline acetyltransferase in the mouse brain under conditions simulating the handling of human autopsy material . Journal of Neurochemistry 31, 381-3 . 9 . Wunner, W . H ., Larson, J . K ., Dietzschold, B . & Smith, C . L. (1988). The molecular biology of rabies viruses . Reviews of Infectious Diseases 10 (Suppl 4), 5771-5784 . 10 . Wunner, W . H . (1991) . The chemical composition and molecular structure of rabies viruses . In The Natural History of Rabies . ( Baer, G . M ., ed .) 2nd edn, pp . 31-67 . Boca Raton, Florida : CRC Press . 11 . Taylor, C . R., Carter, G . I ., Crow, T. J . et al. (1986) . Recovery and measurement of specific RNA species from postmortem brain tissue : A general reduction in Alzheimer's disease detected by molecular hybridization . Experimental and Molecular Pathology 44, 111-6 . 12 . Morrison, M . R . & Griffin, W . S .T. (1981) . The isolation and in vitro translation of undegraded messenger RNAs
Effects of post-mortem autolysis on rabies virus RNA
from
human postmortem
brain .
Analytical
Bio-
chemistry 113, 318-24.
13 . Guillemette, J . G., Wong, L ., McLachlan, D . R . C . & Lewis, P . N . (1986) . Characterization of messenger RNA from the cerebral cortex of control and Alzheimerafflicted brain . Journal of Neurochemistry 47, 987-97 . 14 . Perrett, C . W., Marchbanks, R . M. & Whatley, S . A . (1988) . Characterisation of messenger RNA extracted post-mortem from the brains of schizophrenic, depressed and control subjects . Journal of Neurology, Neurosurgery, and Psychiatry 51, 325-31 . 15 . Johnson, S . A ., Morgan, D . G . & Finch, C . E . (1986) .
235
Extensive postmortem stability of RNA from rat and human brain . Journal of Neuroscience Research 16, 267-80. 16 . Ermine, A ., Tordo, N . & Tsiang, H . (1988). Rapid diagnosis of rabies infection by means of a dot hybridization assay . Molecular and Cellular Probes 2, 75-82 . 17 . Hattwick, M . A . W . (1974). Human rabies . Public Health Review 3, 229-74 . 18 . Perry, E . K ., Perry, R . H . & Tomlinson, B . E . (1982) . The influence of agonal status on some neurochemical activities of postmortem human brain tissue. Neuroscience Letters 29, 303-7.
