Comparative clinical, virological and pathological characterization of equine rotavirus A G3P[12] and G14P[12] infection in neonatal mice

Abstract

Group A rotavirus (RVA) infections are a leading cause of neonatal diarrhoea in foals. Neonatal mice could serve as a useful tool to study the pathogenesis of equine RVA (ERVA) as well as a preclinical model for assessment of vaccine efficacy. This study aimed to comparatively evaluate the clinical, virological and pathological features of ERVA G3P[12] and G14P[12] infection in neonatal mice and compare them with porcine OSU G5P[7] and bovine UK G6P[5] RVA reference strains. Neonatal mice orally inoculated with equine, bovine and porcine RVA developed short-lived diarrhoea at variable rates, G14P[12] (61%) and G3P[12] (88%). Viral replication kinetics for all strains were characterized by a gradual decline in viral load to levels below the limit of detection by 72-96 h post-infection (hpi), in line with the reduction in the number of infected enterocytes demonstrated via RNAscope^® in situ hybridization. Importantly, the clinical and viral replication kinetics correlated with significant microscopic intestinal alterations characterized by enterocyte vacuolation, scalloping and hyperplasia with a peak occurring at 48 hpi and persisting until at least 96 hpi. Overall, neonatal mice develop a disease phenotype of short duration following infection with equine, porcine and bovine RVA strains characterized by diarrhoea and pronounced histological alterations in the intestinal villi. The limited intestinal viral replication is likely associated with host restriction. The clinical and pathological phenotypes developed by neonatal mice following experimental infection could serve as a preclinical tool to assess vaccine efficacy and for pathogenesis studies involving RVA of equine, porcine and bovine origin.

Keywords: G14P[12]; G3P[12]; bovine rotavirus A; equine rotavirus A; neonatal mice; porcine rotavirus A; rotavirus A; rotavirus tropism.

Fig. 1.. Experimental design, disease manifestation and viral load assessment following ERVA G3P[12], ERVA G14P[12], PRVA G5P[7] and BRVA G6P[5] infection in neonatal mice. (a) Overview of the experimental design. Four-day-old neonatal mice derived from timed-pregnant BALB/c mice (E17) were orally infected with 100 µl containing 1×10⁷ TCID₅₀ of ERVA G3P[12], ERVA G14P[12], PRVA G5P[7] or BRVA G6P[5] and monitored for up to 96 hpi. Sampling timepoints included 12, 24, 48, 72 and 96 hpi. (b) Neonatal BALB/c mouse exhibiting yellowish loose diarrhoea, marked for identification. (c) A variable proportion of infected mice developed diarrhoea. (d) Viral genomic copy numbers per mg of intestine following infection. Statistical comparison of viral genome copy numbers per mg of intestine across RVA strains at multiple time points revealed significant differences (Kruskal–Wallis test, P<0.05). Only at 48 hpi, ERVA G14P[12] MCBI exhibited significantly higher viral genomic copy number per mg compared to PRVA G5P[7] OSU and BRVA G6P[5] UK (Dunn post-hoc test, *P values=0.0322 and 0.0294).

Fig. 2.. Temporal dynamics of intestinal lesions following infection of neonatal mice with ERVA G3P[12], ERVA G14P[12], PRVA G5P[7] and BRVA G6P[5]. Cumulative scores were determined following analysis of six histological parameters (villus scalloping, lamina propria alterations, enterocyte vacuolation, presence of neutrophils, intraluminal cell debris and crypt hyperplasia). Mock-infected mice (control group) are depicted in orange. Histological alterations reached their maximum severity at 48 hpi with no significant differences between RVA strains across experimental timepoints (P>0.05). Histological lesions declined by 96 hpi but did not return to baseline values. Each bar represents the mean for lesion severity at a given time point and error bars indicating sd.

Fig. 3.. Temporal progression of individual histological intestinal parameters evaluated following infection of neonatal mice with ERVA G3P[12], ERVA G14P[12], PRVA G5P[7] and BRVA G6P[5]. Panels show lesion severity or presence/absence for six distinct histological parameters: (a) villus scalloping, (b) lamina propria integrity, (c) enterocyte vacuolation, (d) neutrophil infiltration (0=no; 1=yes), (e) intraluminal cell debris (0=no; 1=yes) and (f) crypt hyperplasia. Each bar represents the mean of the parameter, and the error bar indicates the sd.

Fig. 4.. Histological lesions in the small intestine of infected neonatal mice. Image panel depicts (a–e) mock-infected, (f–j) ERVA G3P[12]-infected, (k–o) ERVA G14P[12]-infected, (p–t) PRVA G5P[7]-infected and (u–y) BRVA G6P[5]-infected neonatal mice at 12, 24, 48, 72 and 96 hpi. Changes at 12 hpi included scalloping [e.g. p (inset)] and intraluminal cell debris [e.g. k (arrow and inset)] that evolved to villus blunting (e.g. l) and enterocyte vacuolation [e.g. l, q, v (inset)] by 24 hpi. Prominent vacuolation was evident at 48 and 72 hpi [h, i, m, n, r, s, w, x (arrows)] as well as crypt hyperplasia (h, inset). At 96 hpi, crypt hyperplasia remains evident (e.g. t and y) with sporadic enterocyte vacuolation and transmigrating neutrophils (t, inset). H and E. Magnification 200X.

Fig. 5.. In situ detection of rotavirus RNA at 12, 24 and 48 hpi in the small intestine following infection of neonatal mice with ERVA G3P[12] H2, ERVA G14P[12] MCBI, PRVA G5P[7] OSU and BRVA G6P[5] UK. The bright red punctate to confluent staining represents hybridized viral RNA within infected epithelial cells (black arrows). (a), (d), (g) and (j) At 12 hpi, robust ISH signals were visible in enterocytes, indicating early viral replication. (b), (e), (h) and (k) By 24 hpi, there is a pronounced reduction in the number of infected enterocytes and in the abundance of the RNA signal. (c), (f), (i) and (l) By 48 hpi, viral RNA was undetectable by ISH, suggesting viral clearance. Magnification 400X.