Viral Load and Cell Tropism During Early Latent Equid Herpesvirus 1 Infection Differ Over Time in Lymphoid and Neural Tissue Samples From Experimentally Infected Horses

Abstract

Upper respiratory tract infections with Equid Herpesvirus 1 (EHV-1) typically result in a peripheral blood mononuclear cell-associated viremia, which can lead to vasculopathy in the central nervous system. Primary EHV-1 infection also likely establishes latency in trigeminal ganglia (TG) via retrograde axonal transport and in respiratory tract-associated lymphatic tissue. However, latency establishment and reactivation are poorly understood. To characterize the pathogenesis of EHV-1 latency establishment and maintenance, two separate groups of yearling horses were experimentally infected intranasally with EHV-1, strain Ab4, and euthanized 30 days post infection (dpi), (n = 9) and 70 dpi (n = 6). During necropsy, TG, sympathetic trunk (ST), retropharyngeal and mesenteric lymph nodes (RLn, MesLn) and kidney samples were collected. Viral DNA was detected by quantitative PCR (qPCR) in TG, ST, RLn, and MesLn samples in horses 30 and 70 dpi. The number of positive TG, RLn and MesLn samples was reduced when comparing horses 30 and 70 dpi and the viral copy number in TG and RLn significantly declined from 30 to 70 dpi. EHV-1 late gene glycoprotein B reverse transcriptase PCR and IHC results for viral protein were consistently negative, thus lytic replication was excluded in the present study. Mild inflammation could be detected in all neural tissue samples and inflammatory infiltrates mainly consisted of CD3+ T-lymphocytes (T-cells), frequently localized in close proximity to neuronal cell bodies. To identify latently infected cell types, in situ hybridization (ISH, RNAScope®) detecting viral DNA was used on selected qPCR- positive neural tissue sections. In ganglia 30 dpi, EHV-1 ISH signal was located in the neurons of TG and ST, but also in non-neuronal support or interstitial cells surrounding the neuron. In contrast, distinct EHV-1 signal could only be observed in neurons of TG 70 dpi. Overall, detection of latent EHV-1 in abdominal tissue samples and non-neuronal cell localization suggests, that EHV-1 uses T-cells during viremia as alternative route toward latency locations in addition to retrograde neuronal transport. We therefore hypothesize that EHV-1 follows the same latency pathways as its close relative human pathogen Varicella Zoster Virus.

Keywords: Alphaherpesviruses; EHV-1; horses; latency; lymphocytes; pathogenesis; trigeminal ganglia.

Figure 1

Significant differences (**P < 0.01) in viral copy numbers in (A) trigeminal ganglia and (B) retropharyngeal lymph nodes at 30 dpi compared to 70 dpi. Viral copy numbers in (C) sympathetic trunk and (D) mesenteric lymph nodes at 30 dpi compared to 70 dpi. Line indicates the median.

Figure 2

(A) Trigeminal Ganglion 30 dpi: Moderate infiltration of inflammatory cells (white arrow) and satellitosis (arrowhead); (B) Sympathetic trunk ganglion 30 dpi: Nageotte's body (N) as sign of neuronal degeneration; Bars = 50 μm.

Figure 3

Trigeminal Ganglion 30 dpi, IHC for CD3 and CD20, DAB with Mayer's hemalum counterstaining: (A) CD3+ T-cell infiltration in vicinity of blood vessel (BV); (B) CD3+ T-cells penetrate neuron-satellite sheet (arrowhead); (C) localized infiltrates of CD20+ B-cells associated with non-labeling T-cells; bars = 50 μm.

Figure 4

In situ hybridization for EHV-1 gB. (A) Positive control with positive labeling (arrowheads); (B) Positive control tissue with no signal using an EHV-1 scrambled probe; (C) TG from a EHV-1 qPCR negative horse lacks positive ISH signal; (D) EHV-1 qPCR positive TG 30 dpi with strong signal in non-neuronal cells (arrowheads); (E) EHV-1 qPCR positive TG 30 dpi with strong in non-neuronal cells (white arrowheads) and nucleus of the neuron (black arrowhead); (F) EHV-1 positive TG 70 dpi with positive signal in the nucleus of the neuron (arrowhead). Bars = 50 μm.