Spatiotemporal dynamics of alphaherpesviral latency and reactivation in the murine central nervous system

Abstract

Alphaherpesviruses, including Herpes Simplex Virus 1 (HSV-1) and Pseudorabies Virus (PrV), establish lifelong latency in the nervous system and can cause recurrent disease. While latency has classically been attributed to peripheral sensory ganglia, accumulating evidence indicates that the central nervous system (CNS) also act as a relevant reservoir for viral latency and reactivation. Here, we investigated the CNS as a site of latency using the attenuated mutant PrV-{Delta}UL21/US3{Delta}kin which reproduces key features of herpes simplex encephalitis (HSE) in female CD1 mice. We mapped brain regions permissive to alphaherpesviral latency and analyzed the temporal dynamics of viral transcription, histopathology, and host clinical and immune responses. Following intranasal inoculation, mice were analyzed at 11 to 14, 21, 28, 42, 105, and 190 days post infection (dpi). To assess the potential of reactivation, a subset received cyclophosphamide/dexamethasone at 170 dpi. Viral transcripts were detected by RNAscope in situ hybridization and RT-qPCR targeting the lytic gene UL19 and the latency-associated transcript (LAT). Histopathological analyses included hematoxylin and eosin (H&E) staining and immunohistochemistry for CD3, Iba1, GFAP, and cleaved caspase-3. Major capsid protein (UL19) expression displayed marked regional and temporal heterogeneity, with prominent signals in mesiotemporal structures (piriform cortex, hippocampus, entorhinal cortex), coinciding with pronounced T-cell infiltration. LAT expression remained overall low, with a transient peak during the acute infection (11-14 dpi). RT-qPCR confirmed high viral transcript levels for both UL19 and LAT in mesiotemporal regions during early infection, while LAT expression returned to baseline levels thereafter. Histopathology demonstrated a transition from acute necrotizing meningoencephalitis to prolonged or recurrent low-grade inflammation, accompanied by glial activation and localized apoptosis. Notably, UL19 expression strongly correlated with CD3+ T-cell infiltration, particularly at 42 dpi. These findings define the spatiotemporal interplay between viral transcriptional activity and neuroinflammation and identify selected CNS regions as reservoirs for latent or recurrent alphaherpesvirus infection. Author SummaryAlphaherpesviruses are pathogens that not only cause acute disease but also establish lifelong latency in the nervous system. Under certain conditions, they can reactivate and trigger recurrent disease. Using a genetically modified pseudorabies virus in mice, we mapped how latent and lytic phases occur within the brain over several months. By combining molecular, histological, and clinical analyses, we show that specific brain regions act as long-term viral reservoirs, where signs of inflammation and viral activity can reappear long after the initial infection. These findings provide new insights into how alphaherpesviruses persist in the central nervous system and suggest that recurrent or subclinical reactivation may contribute to long-term neurological complications, including those resembling human herpes simplex encephalitis.