Clinical manifestations after infection. The kittens in the RHDV-S group showed no obvious clinical symptoms after inoculation, and they were sacrificed at 72 hours post-infection(hpi). Other infected rabbits died within 3 days after inoculation. The death time of rabbits in the RHDV2-S group was the shortest, and all kittens died within 18 hours. All the dead rabbits showed typical clinical symptoms of RHD. The specific manifestations included: depression, loss of appetite, sudden death, violent struggle before death, angular arch retraction, and oral bleeding in some rabbits.
Temperature changes preceding death following RHDV/RHDV2 infection. After RHDV infection in rabbits, pyrexia peaked on average 6 hours prior to endpoint (range 4 ~ 12 hours before the endpoint) in both adult rabbits and subadult rabbits. The body temperature of young rabbits increased within 24 hours and then tended to be stable. The surviving kittens in the infection experiment were killed manually at 72 hours to determine visceral viral load.
After RHDV2 infection in rabbits, pyrexia peaked on average 6 hours before endpoint (range 4 ~ 10h before the endpoint) in all groups. Unlike RHDV infection, RHDV2 was lethal to kittens, who died the fastest and had the fastest rise in body temperature after infection with RHDV2.
Kinetics of viremia after RHDV infection. The kinetics of viremia after RHDV infection as shown in Fig. 3. The broken red line represents the dynamic change in virus copies in blood. The virus copies in the blood of kittens began to rise at 6 hpi, reached the highest at 24 hpi, and then continued to decline. The virus copies in the blood of other rabbits increased rapidly within 6 hpi, then slowed down.
The black broken line represents the dynamic change of virus copy number in feces. The virus copy number in the feces of kittens and subadult rabbits continued to increase within 9 hpi, and then tended to be stable. The virus copy number in feces of adult rabbits increased within 3 hpi, then tended to be stable, then increased again after 12 hpi.
The broken blue line represents the dynamic change of virus copy number in saliva. The number of virus copies in the saliva of kittens increased within 9 hpi, then became flat, and began to decrease after 48 hpi. At 72 hpi, RHDV could not be detected. The virus copies in the saliva of subadult rabbits and adult rabbits increased first and tended to be stable.
Kinetics of viremia after RHDV2 infection. The kinetics of viremia after RHDV2 infection as shown in Fig. 4. The broken red line represents the dynamic change in virus copies in blood. The virus copies in all rabbits' blood increased rapidly, but the trend was slower in adult rabbits, and the time of adult rabbits’ death was later.
The black broken line represents the dynamic change of virus copy number in feces. The virus copies in feces of all rabbits increased within 6 hpi, and then stabilized.
The broken blue line represents the dynamic change of virus copy number in saliva. After 6 hpi, the virus copies in the saliva of all rabbits increased to a stable level. The virus was detected in the saliva of young rabbits within 3 hpi, while it was detected in the saliva of subadult and adult rabbits after 3 hpi.
Visceral viral load and pathological score. The results of HE staining in rabbit viscera were shown in Supplementary Figure S1-S7. Combined with the score table of pathological sections of rabbits in the challenge group(Supplementary Table S1 ~ S5) and the distribution of visceral viral load of rabbits in the challenge group(Supplementary Table S6 ~ S7), the bar chart of visceral viral load and a pathological score of rabbits in challenge group was drawn.
After RHDV challenge. The results showed that after RHDV challenge, there was a trend between visceral viral load and lesion scores. No apparent lesions were found in the heart of rabbits in all experimental groups, and the virus load was the lowest. The liver, spleen and lung tissue virus load of adult rabbits higher than in kittens and subadult rabbits, and the lesion score was higher than in kittens and subadult rabbits. There were no significant differences in renal viral load and renal lesion scores among rabbits.
After RHDV2 challenge. The results showed a particular trend in the virus loads and lesion scores of viscera after RHDV2 challenge. Compared with the other groups' hearts, the adult rabbits' hearts had the lowest virus load and no lesions. The virus load of the liver in kittens was higher than in subadult and adult rabbits, and the lesion score was the highest. The virus load of the spleen in adult rabbits was much higher than in kittens and subadult rabbits, and the lesion score was also higher. The lung virus load in adult rabbits was higher than in kittens and subadult rabbits, and the lesion score was also the highest. No apparent lesions were found in the kidneys of all rabbits in RHDV2 challenge group.