Report on simultaneous infection of rabbit haemorrhagic disease virus type 1 and type 2 in kits in China

Abstract Diagnosis of the causes of rapid mortality in on rabbits was carried out on the basis of epidemiological and pathological profiling, using RT-PCR testing, gene sequencing and phylogenetic tree construction. In this experiment, samples were collected from two rabbit farms in Guizhou and Henan Provinces, China. Then the total RNA of liver tissue was extracted by Trizol method for RT-PCR amplification. The results showed that the specific target band was observed at 829 bp of RHDV2 and 591 bp of RHDV by agarose gel electrophoresis. Two different RT-PCR products amplified from one rabbit liver sample were named GZ-RHDV and GZ-RHDV2, respectively, and the other farm was named HN-RHDV and HN-RHDV2. Subsequently, four RT-PCR products amplified from two rabbit liver samples from different farms were selected for gene sequencing respectively, and the gene sequences were uploaded to NCBI for blast analysis. Finally, MEGA-7 software was used to construct the phylogenetic tree. The measured gene sequences were analysed by blast analysis and the amplification products in the liver samples showed a high degree of homology with domestic virus isolates. Among them, GZ-RHDV and HN-RHDV shared 98.50% and 98.69% homology with the Genbank accession number (MK814815.1), GZ-RHDV2 shared 99.35% homology with the Genbank accession number (OQ570963.1), and HN-RHDV2 shared 98.84% homology with the Genbank accession number (OQ570961.1). The above results confirmed that the cause of this mass mortality in rabbits on both farms was co-infection with RHDV and RHDV2. The sequenced liver samples from farms in Guizhou Province were selected to prepare virus suspension, and 60 day old rabbits immunized with RHDV vaccine were subcutaneously injected into the neck to identify the virulence of the virus. After 22 h, the infected rabbits developed typical clinical symptoms. For example, typical blood retention occurred in the mouth and nose, convulsions, opisthotonos, Mucoid secretion of anus, bleeding in liver, lung and heart, congestion and swelling in spleen were found during autopsy. The results showed that the classical vaccine had no protective effect on the mixed infection strain.