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.

Transcriptomic Profiling of Equine and Viral Genes in Peripheral Blood Mononuclear Cells in Horses during Equine Herpesvirus 1 Infection

“…It is possible that this depth of next-generation sequencing is more sensitive than our qPCR assay, and it is also possible that viral transcripts are more abundant in samples than latent genomic DNA. While the trigeminal ganglia is the trademark site for EHV-1 latency, many studies have reported EHV-1 latency in lymphoid tissues and PBMCs as well as in additional ganglia and lymphoid tissues [ 3 , 4 , 5 , 6 , 7 ]. It is presumed that most horses become infected with EHV-1 at a very young age (days to weeks old) [ 81 , 82 , 83 , 84 ].…”

“…The classical theory of herpesvirus biology describes sensory ganglia as the primary target for latency of alphaherpesviruses and lymphocytes as the primary latency site for gammaherpesviruses [ 1 ]. While several reports have identified lymphoid tissues as a site of latency for EHV-1, genome detection in circulating PBMC via qPCR is often negative in non-clinically affected animals [ 3 , 4 , 5 , 6 , 7 , 96 ]. In contrast, EHV-2 and EHV-5 can be routinely detected in PBMC samples of healthy animals [ 96 , 97 ].…”

“…Alphaherpesviruses are known for their rapid lytic replication in many cell types [ 2 ]. Alphaherpesviruses typically (though not exclusively) establish latency in the sensory ganglia of their host; however, EHV-1 is also known to establish latency in lymphoid tissues [ 3 , 4 , 5 , 6 , 7 ]. On the other hand, gammaherpesviruses are more restrictive in their cell tropism and are known to establish latency primarily in lymphocytes as well as for their slow replication cycle [ 1 , 8 ].…”

“…This allows the virus to effectively evade immune surveillance within the PBMCs while being transported to the secondary sites of infection. Furthermore, it is likely that viremia is an important step in the establishment of latency of EHV-1 in lymphoid tissues [ 7 ].…”

The Pathogenesis and Immune Evasive Mechanisms of Equine Herpesvirus Type 1

“…Latent EHV-1 was detected by PCR and recovered by co-cultivation from lymphoid tissues draining the respiratory tract of experimentally inoculated ponies ( Welch et al, 1992 ; Kydd et al, 1994 ; Slater et al, 1994a ). Additional studies stating that EHV-1 establishes latency in lymphoid tissues, failed to conclusively demonstrate the presence of LATs in these tissues ( Pusterla et al, 2010 , 2012 ; Goehring, 2017 ; Giessler et al, 2020 ). Finally, circulating T lymphocytes have been defined as a predominant site of EHV-1 latency.…”