Genomic Analysis And Pathogenicity Study Of A Rabbit Haemorrhagic Disease Type 2 Virus Strain

Rabbit Haemorrhagic Disease Virus Type 2(RHDV2),first isolated in China in 2020,can infect rabbits of different ages and shows a high morbidity and mortality rate especially in young rabbits.Clinically,the disease is characterized by depression,anorexia,severe hypothermia,respiratory distress,unresponsiveness,bradycardia,cardiac arrhythmia,frothy red blood coming from the mouth and nose of sick rabbits,and a loose anus with yellow dilute feces.This study was based on experiments on a suspected case of RHDV2 in a rabbit farm in Shandong Province.Through a series of studies,it was identified as RHDV2,and whole genome sequence amplification analysis and preliminary pathogenicity experiments were conducted,and the following results were obtained.1.Identification of RHDV2 in clinical disease materialIn order to identify the pathogenic agent of rabbit mortality on this farm,liver suspensions were first subjected to a hemagglutination test,which showed its hemagglutinating properties.PCR amplification was then performed using typing primers for RHDV,and agarose gel electrophoresis observed a specific target band at 480 bp corresponding to RHDV2,while no amplified band was observed at 193 bp corresponding to RHDV1.Sequence alignment analysis confirmed that the target fragment obtained from the disease material was the VP60 partial sequence of RHDV2,which was highly homologous to the domestic RHDV2 strain.The RHDV2 strain identified in this material was named SD-2021.Additional PCR amplification using primers for Pasteurella rabbitis identification did not amplify a specific band.2.Amplification and analysis of the whole gene sequence of strain SD-2021In order to understand the genomic information of this strain,eight pairs of whole sequence amplification primers were designed based on the sequence of Sichuan RHDV2 strain SC2020/0401(MT586027),and SD-2021 was amplified by fragmentation.The amplification results showed that the size of the eight fragments amplified from the liver was consistent with the expected fragment size.The full-length gene sequence of 7443 bp was obtained by splicing using DNAStar,followed by phylogenetic tree and homology analysis using MEGA11.SD-2021 was found to have 88.03% genome-wide homology,98.91% VP60 nucleotide homology and 84.46% non-structural nucleotide homology with Sichuan SC2020/0401(MT586027)and CHN/SC2020(MT434995);phylogenetic tree analysis revealed that the isolated SD-2021 strains were in The phylogenetic tree analysis revealed that the isolated SD-2021 strain formed an independent branch in RHDV2 and classical RHD.In response to the low genome-wide homology with the Sichuan strain,recombination analysis with RDP and Simpolt software confirmed that a recombination event occurred in SD-2021 with a recombination breakpoint at 5266 bp,and its primary parental strain was RHDV1/Triptis(EF558583)and the secondary parents RHDV2/SC2020/0401(MT586027).3.Pathogenicity study of SD-2021 strainThe liver tissue suspension was injected subcutaneously into 30-day-old rabbits for attack.After attack,typical clinical symptoms such as bleeding from the mouth and nose,convulsions and corns were observed,and hemorrhage was found in the liver,lungs and heart on autopsy,and the spleen was enlarged at the margin.The pathological section could be observed under a 20× field of view to show disorganized liver tissue structure,with obvious diffuse necrosis of hepatocytes in the central veins,confluent areas and liver parenchyma.Fluorescence quantification of the heart,liver,spleen,lung,kidney,duodenum,trachea and feces of their dead rabbits revealed that the highest viral content was in the liver tissue,while the rest of the organs contained virus,and some of their feces were detectable and some were not.The results were 104.6-104.8.When the same method was used to attack rabbits at 30,40 and 60 days of age,it was found that rabbits at younger ages were more sensitive to the virus.In summary,this study found for the first time a recombinant strain of RHDV2 in China,indicating that RHDV2 is constantly mutating and has genetic diversity.Moreover,the in vivo replication test of its recombinant strain confirmed its virulence to rabbits,which provides data to support the clinical prevention and control of RHDV2.