Isolation, Identification And Reverse Genetic Of Mink Reovirus

Reovirus was first isolated during the 1950 s and the first double stranded RNA virus was described. It was isolated mainly from the respiratory or intestinal tract with minimal or no associated disease symptoms, so it was called respiratory, intestinal and orphan virus, referred to as reovirus.Mammalian Orthoreoviruses(MRVs) belong to the family Reoviridae under genus Orthoreovirus. MRVs are spherical, icosahedral, nonenveloped particles. Virions are formed by two concentric capsid protein shells that surround and protect a genome of 10 segmented double-stranded RNA(dsRNA) that are divided into three groups, large(L1~L3), medium(M1~M3) and small(S1~S4), which encode 8 structural proteins and 3 nonstructural proteins. Four major reovirus serotypes have been recognized so far(type 1 Lang, type 2 Jones, type 3 Dearing, type 4 Ndelle).To date, reports on the isolation of reovirus from minks are limited. In 1975, the first report of the isolation of reovirus from mink was described in Germany. In 1992, Liu et al. also reported the isolation of a reovirus from the feces of minks with diarrhea in China. In 2013, Lian et al. reported firstly a complete genome of a type 1 reovirus of MRV1 HB-A. Although reovirus infection is no obvious symptoms or mild symptoms, virus mixed infection can aggravate the disease and result in mink growth retardation, unkempt fur. Thereby the quality of mink fur was bad and breeding industry lost seriously.Reverse genetics technology has been widely applied in virology research, which greatly promoted the development of virology. In classical genetics, the specific genes in an organism were deduced from observations of the phenotype of the organism. Reverse genetics is a term coined to describe processes where information flows in the opposite direction. The changes was introduced directly into the complementary DNA(cDNA) used to generate infectious RNA virus, in order to study gene replication, expression and regulation mechanism, virus and host interactions, antiviral strategies, gene therapy, the construction of a new virus vector to express foreign genes and new vaccines. Reverse genetics technology was an useful method in the molecular virology. The complexity of reovirus genome hampered the establishment of reverse genetic system and restricted the research on various aspects of the virus.There was an outbreak of disease with diarrhea in a mink farm in Shandong in 2014. The intestinal contents were inoculated into Vero and BHK cells. The cells were passaged every 3 days and clearly observed for viral cytopathic effect(CPE) in the third passage and cells were rounded and detached. The supernatants of medium containing viral particles were observed by negative staining electron microscopy. The observed virus particles were nonenveloped and isometric with a double-layered capsid and were proximately 75 nm in diameter. RT-PCR and BLAST showed that the virus was mammalian orthoreovirus, referred to as MRV3 SD-14. The TCID50 was 105.625 TCID50/mL and 105 TCID50/mL in BHK-21 and Vero. It didn’t display the capacity to agglutinate human type O erythrocytes and chicken, guinea pig, cattle, rabbit red blood cells at 4 ℃, room temperature and 37 ℃. SD-14 strain was inoculated into suckling mice, which was not pathogenic to mice. The complete genome sequence of 10 segments of the virus was determined by special primers and sequenced by Comate Bioscience Co., Ltd., Changchun, China. The data were submitted into the GenBank database(Accession numbers KT224504-KT224513). The nucleotide sequences of each segment were compared with other orthoreoviruses including the prototype MRVs used DNAMAN. The results showed that four segments(L1, L3, M3, S3) of MRV3 SD-14 were closely related to human reovirus(MRV2Tou05) and the sequences concidence rates were 97.40 %, 96.92 %, 97.09 % and 97.20 %, respectively. The S2 segment of MRV3 SD-14 was most similar to type 1 Lang(96.17 % identity). The other five segments shared high sequence similarity with swine-originated MRVs strains GD-1 and SC-A which were isolated in Guangdong and Sichuan Province, China(sequence coincidence rates were from 95.53 % to 98.66 %). Meanwhile, MRV3 SD-14 showed high similarity with MRV1 HB-A which was also isolated from mink in 2011 in Hebei, China, except for S1 segment. Each segment was also determined and analyzed phylogenetically by MEGA. In conclusion, we supposed that MRV3 SD-14 was reassorted between human, swine and/or mink strains. S1 segment had a stop codon mutation at codon 246, which maybe result in a truncated σ1 protein and affect the viral characteristics. We isolated a novel natural mutant and reassortant type 3 reovirus(MRV3 SD-14) from mink. SD-14 strain inriched reovirus gene pool and biological characteristics, which was important to study reovirus prevalence and evolution in mink groups.In order to learn reovirus infection status of mink groups, microneutralization test was performed to determine reovirus seroprevalence rate. 274 serum samples were collected from mink farms in Shandong, Hebei and Jilin. The seroprevalence rate in mink groups were 92.5 %, 82.1 % and 91.7 % respectively in above areas. The results showed that reovirus infection rate was very high in mink groups.In order to study the protein function and pathogenic mechanism of virus, we hoped that we could establish a reverse genetic system of reovirus. In this study, the whole genome sequences of the HB-A strain was confirmed. The cDNA sequences of virus was inserted into expression cassette which containing T7 promoter, HDV ribozyme and T7 terminater. The 10 recombinant plasmids pcDNA-T7-L1, pcDNA-T7-L2, pcDNA-T7-L3, pcDNA-T7-M1, pcDNA-T7-M2, pcDNA-T7-M3, pcDNA-T7-S1, pcDNA-T7-S2, pcDNA-T7-S3 and pcDNA-T7-S4 were obtained. The Tâ†'C substitution at position 2193 in L1 was used for marker rescue. The start codon of the σ1s open reading frame was changed(AUG to ACG) which was used to construct rsHB-A σ1s-null. BHK-T7 cell was contransfected with 10 recombinant plasmids and incubated at 37 ℃ for 3~5 days.We reported here the first case of a natural mutant and reassortant serotype 3 reovirus from mink in China, known as MRV3 SD-14. The main biological characteristics of virus were identified. Reovirus infection rate in mink groups was confirmed by serological survey and the result showed that reovirus infection was common in mink groups. A preliminary study of reverse genetic of HB-A strains was established, which could lay the foundation for studying virus infection, recombinant, pathogenic mechanism.