Equid herpesvirus 1 (EHV-1) is a pathogen that causes myeloencephalopathy in horses and, occasionally, in other species such as mice. As few EHV-1 strains have been identified as highly virulent to this species, despite mouse models being used to understand EHV-1 pathogenesis, a study by Mesquita et al. (2021) aimed to evaluate the pathogenesis of two neurovirulent strains of the disease in mice. The study also characterised the inflammatory cells, the expression of chemokines and the apoptosis marker caspase-3 in the brains of the infected individuals.
In our latest NationWide laboratories journal club, Marvin Firth, BVSC (Hons), DIPRCPath, DIPFMS, AFHEA, MRSB, MRCVS, dives into the research conducted by Mesquita et al. to discover the pathogenesis of EHV-1 infections in the central nervous systems of mice.
Background
We often think of EHV-1 as causing severe clinical manifestations in horses – this includes myeloencephalopathy, an often fatal neurological disorder characterised by changes in the brain and spinal cord. What is intriguing about the disease, however, is that it can also cause disease in other species. EHV-1 can cause fatal meningoencephalitis in the black bear, Thompson’s gazelles, mice and others, including some critically endangered species.
Mouse models have been used to study the pathogenesis of EHV-1 infections in the past. Some papers found a strong tropism for respiratory disease, while others demonstrated a fatal central nervous system infection. As with equids, mice can experience the spectrum of EHV-1 disease from different strains of herpesvirus.
In the study, the authors focused on two strains prevalent in neurological tissue: A4/72 and A9/92. Despite previous classification as non-neuropathic, these strains often appeared to cause high mortality rates in animal models (with the Ab4 strain causing 50 percent mortality at three days post-inoculation (DPI) and A4/72 and A9/92 extremely neurovirulent at 100 percent mortality within three to four DPI).
The hope was to demonstrate the mechanism underlying the neuropathology experienced in mice as a result of these viral infections and provide a new model for EHV-1.
Method
Thirty-two male mice (five to six weeks old) were inoculated intranasally with the two strains of EHV-1 and were evaluated on each of the first three days post-inoculation.
Histological (with haematoxylin-eosin (HE)) and immunohistochemical analyses were then conducted on four mice inoculated with each strain at each time point (24/32). The brains of four infected mice from each group were also analysed with electron microscopy (4/32), and four mice were in a control group (4/32).
Each day, the mice were weighed and examined for neurological signs, including seizures, hindlimb paralysis and recumbency, as well as for alternate signs like ruffled hair and hunched posture.
| “All experimental procedures were approved and carried out according to the Ethics Committee for the Use of Animals” of the animal facility of the department of pathology, School of Veterinary Medicine and Animal Science at the University of Sao Paulo, Brazil (Mesquita et al., 2021). |
Results
This paper revealed numerous interesting aspects of the pathophysiology of these EHV-1 strains in terms of the tissue changes visible under the microscope and via advanced imaging techniques (as seen in Figure 1).
Electron microscopy
Electron microscopy allows us to visualise the membrane-bound virus capsules within the brain and nasal tissue of infected mice (Figure 1; 5 and 6). These extracellular viral nucleocapsids were numerous and frequently organised in clusters, but most viral particles were naked, with enveloped virions detected scarcely.
This modality also revealed large numbers of degenerative and necrotic (neuron) cells with:
- Clumping and dissolution of nuclear chromatin in cells
- Fragmentation of nuclear and cytoplasmic membranes
- Dissolution and vacuolation of the cytoplasm, which had degenerated cells (especially mitochondria)
- Multifocal disruption and vacuolation of the neuropil
- Decreased astrocytes, those present containing markedly swollen foot processes
Neurons in less affected areas exhibited normal morphology, characterised by numerous organelles and cytoplasmic prolongation consistent with dendrites and axons, of which the neuropil was still intact.
Ultimately, electron microscopy revealed numerous viral nucleocapsids and fewer enveloped virions in degenerate and necrotic neurons and the surrounding neuropil. The olfactory bulb (OB) and ventral portion of the brain and meninges were most affected.
Histopathology
The authors also visualised the areas of the brain and nasal epithelium damaged by exposure to the EHV-1 strains via histopathology and immunohistochemistry to determine the nature, intensity and distribution of the lesions. At 3 DPI, all mice infected with an EHV-1 strain had brain lesions.
HE showed circulating blood cells entering the perivascular space, causing the release of chemicals that can cause damage to neurons and the supporting structures of the brain.
Other interesting finds included:
- Multifocal to coalescing areas with loss of neuropil and vacuolation
- Severe and multifocal loss of periglomerular cells in the glomerular layer, with pyknotic and karyorrhectic nuclei in the remaining cells
- Multifocal loss of mitral cells, with remaining cells showing shrunken hypereosinophilic cytoplasm and karyorrhectic nuclei
- Disrupted axons extending from the olfactory nerve layer, admixed with cellular debris and oedema
- Severe, multifocal to coalescing loss and cavitation of neuropil adjacent to areas of neuronal necrosis in certain areas of the brain and central nervous system
- Diffusely distended perivascular spaces (oedema), with hypertrophy of endothelial cells and congestion
- Marked loss of ciliated epithelium replaced with amorphous hypereosinophilic material in the nasal cavity
- Epithelial cells with pyknotic or karyorrhectic nuclei frequently sloughed into the lumen of the nasal cavity
Immunohistochemistry
Immunohistochemistry was used to detect and analyse chemokines (CCL5 and CCL2), T lymphocytes and macrophages/microglia. This was included because the authors were aware that certain chemicals (cytokines and chemokines) released during acute inflammation and an increased number of white blood cells (blood leukocytes) in circulation could cause indirect damage to neuroparenchyma, brain cells and the nasal and olfactory bulbs.
The most significant result of immunohistochemistry was the discovery of perivascular cuffing of CD3+ T cells (Figure 2; 17) and Iba-1 cells (Figure 2; 18), with areas concomitantly expressing CCL2 and CCL5 chemokines – the critical regulators of leukocyte trafficking into the central nervous system in viral infections.
Further results included:
- CD3+ cells identified in areas where lymphocytes were not histologically evident (indicating more widespread oedema than previously described)
- Diffuse immunolabelling for Iba-1 in most analysed areas, characterised by activated microglia with a morphology of thickened microglial processes and enlarged and rounded nuclei
- Immunolabelling for CCL5 and CCL2 was more intense in areas with significant histological lesions but also detected in other areas
- Caspase-3 immunolabelling was most present in areas with necrotising lesions
- In areas exhibiting necrosis/degeneration, caspase-3 labelling was granular and in the neuroparenchyma adjacent to necrotic neurons. Where significant histological lesions were not present, immunolabelling was strong, granular, cytoplasmic staining in neurons and cells with glial morphology
Conclusions
Overall, Mesquita et al. found that mice do develop neurological disease when inoculated intranasally with the A4/72 and A9/92 strains of EHV-1, which was invariably fatal by 3 DPI. The mice exhibited severe necrotising, lymphocytic and histiocytic encephalitis with extensive neuronal necrosis, microgliosis and the expression of CCL2, CCL5 and caspase-3.
However, in mice, this neurological disease occurs from a different route to EHV-1 infection in horses. Mice experience changes that cause rhinitis that extends back towards the central nervous system. Horses, on the other hand, express the disease due to entry via lymph nodes and circulation in the bloodstream to the brain.
Take-home message
But what does this all mean? What message should you take home from this study? Well, ultimately, the model presented by Mesquita et al. (2021) could be useful in furthering our understanding of how equine herpesviruses, particularly EHV-1 and EHV-9, could affect our more endangered species, such as rhinos, or even those a little closer to home (eg guinea pigs and hamsters).
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