The pathogenicity comparison of Lagovirus europaeus GI.1 and GI.2 strains in China by using relative quantitative assay

Abstract

Lagovirus europaeus GI.1 belongs to Lagovirus in the Caliciviridae family. GI.1 causes an acute, septic, and highly lethal disease in rabbits. Lagovirus europaeus GI.2, a new variant of GI.1, has caused explosive mortality in rabbits of all ages in Sichuan Province, China. To explore the differences in pathogenicity of rabbits infected with GI.1/GI.2, we investigated the virulence and disease progression of a naturally occurring GI.1/GI.2 in 4-week-old, 13-week-old, and 25-week-old New Zealand White laboratory rabbits after GI.1/GI.2 infection. Objective measures of disease progression were recorded using continuous body-temperature monitoring. We observed the kittens were infected with GI.2 during the most urgent course of the disease, and GI.1 was not lethal to kittens. We found that the target organ of both GI.1 and GI.2 was the liver, but the disease course of the two viruses was differed. Our study enriches the research on the pathogenicity of GI.1 and GI.2 under the same conditions.

Figure 1

Temperature changes preceding death following GI.1 infection. To ensure the data were accurate, 6 researchers measured each rabbit's rectal temperature every three hours after infection, day and night. Mean temperature for timepoint is listed with the standard deviation. Red dotted lines indicate when rabbits were sacrificed/died at different timepoints. (a) Body temperature changes before the adults’ humane endpoint (at 24 hpi); (b) Body temperature changes before the subadults’ humane endpoint (at 36 hpi); (c) Body temperature changes before the kittens were sacrificed (at 72 hpi).

Figure 2

Temperature changes preceding death following GI.2 infection. To ensure the data were accurate, 6 researchers measured each rabbit's rectal temperature every three hours after infection, day and night. Mean temperature for timepoint is listed with the standard deviation. Red dotted lines indicate when rabbits were sacrificed/died at different timepoints. (a) Body temperature changes before the kittens’ humane endpoint (at 18 hpi); (b) Body temperature changes before the subadults’ humane endpoint (at 21 hpi); (c) Body temperature changes before the adults’ humane endpoint (at 30 hpi).

Figure 3

Viral copies in blood, feces, and saliva over time after GI.1 infection. The red, black, and blue broken lines represent the dynamic changes of viral copies in blood, feces, and saliva, respectively. Mean value is listed with the standard deviation. The orange line represent the control group, and the viral copies of samples tested at each timepoint was 0. After GI.1 infection, (a) for kittens, the viral copies in blood and saliva increased first and then decreased, while the viral copies in feces remained stable; while the viral copies in blood, saliva and feces in (b) subadults and (c) adults showed an increasing and then stabilizing trend.

Figure 4

Viral copies in blood, feces, and saliva over time after GI.2 infection. The red, black, and blue broken lines represent the dynamic changes of viral copies in blood, feces, and saliva, respectively. Mean value is listed with the standard deviation. The orange line represent the control group, and the viral copies of samples tested at each timepoint was 0. After GI.2 infection, all rabbits [(a) kittens, (b) subadults, (c) adults] had an increased viral copies in blood, while the viral copies in feces and saliva remained stable after 6 hpi.

Figure 5

Column representation of visceral viral loads and a pathological scores of rabbits infected with GI.1. The viral load was calculated according to the formula 2^−ΔΔCT. The higher the ΔCt value is, the lower the 2^−ΔΔCT value is. The heart with the highest ΔCt value and the lowest expression of GI.1 was selected as Calibrator. Different asterisks indicate significant differences, *(p < 0.05), **(p < 0.01), ***(p < 0.001), ****(p < 0.0001); ns means the difference is not significant (p > 0.05).

Figure 6

Column representation of visceral viral loads and a pathological scores of rabbits infected with GI.2. The viral load was calculated according to the formula 2^−ΔΔCT. The higher the ΔCt value is, the lower the 2^−ΔΔCT value is. The kidney with the highest ΔCt value and the lowest expression of GI.1 was selected as Calibrator. Different asterisks indicate significant differences, *(p < 0.05), **(p < 0.01), ***(p < 0.001), ****(p < 0.0001); ns means the difference is not significant (p > 0.05).