IN-SITU PCR LOCALIZATION OF HERPES-SIMPLEX VIRUS-DNA SEQUENCES IN DISSEMINATED NEONATAL HERPES-ENCEPHALITIS

To more precisely define the role of herpes simplex virus (HSV) in development of nervous system disease in neonates with disseminated infection, an in situ polymerase chain reaction (ISPCR) method was used to detect and localize HSV DNA sequences in paraffin sections of neural and non-neural autopsy tissues. In subregions of adjacent sections corresponding to ISPCR-labeled and unlabeled areas, HSV specificity was verified using solution PCR and Southern blots. In serial sections, ISPCR results were compared to lesions, HSV antigen and, in selected samples, to viral sequence detection by in situ hybridization. By ISPCR, HSV-specific labeling was limited to HSV-infected neonates and experimentally infected mouse controls. ISPCR-labeled cells corresponded to regions that were histologically abnormal and contained HSV antigen or in situ hybridization signal in some foci; in others, labeled cells were in areas with no evident lesions or antigen. Results suggest two routes of HSV spread to the CNS: (i) blood-borne infection, with HSV DNA in splenic lymphocytes, circulating cells, meningeal vessel walls and cells in intraventricular hemorrhage, and (ii) neural spread, with HSV detected in brain stem sensory neurons. In the brain of one neonate surviving acute infection, detection of HSV nucleic acid sequences suggests a latent or persistent viral genome. With other methods, this highly sensitive ISPCR technique permits a more complete definition of HSV infection in these infants and provides new insights into disease mechanisms. Fuller understanding of HSV persistence and recurrent neurological disease in survivors will require further studies using these and other techniques in human tissues and in animal models.