The Study On Properties, Complete Genomic Sequence Analysis, And Molecular Detection Methods Of Pike Fry Rhabdovirus

Pike fry rhabdovirus (PFRV) shares high homology to Spring viraemia of carp virus (SVCV), but has not attached great importance to people. In order to make a more comprehensive understanding of PFRV, and lay the foundation for the future inspection and control of PFRV, its biological characteristics were studied, the complete genome sequence was cloned and sequenced, and three detection methods were developed, including reverse transcription polymerase chain reaction (RT-PCR), real time RT-PCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP).1. The biological characteristics of PFRV. PFRV was inoculated onto the monolayers of Bluegill fry-2 (BF-2), Fathead minnow (FHM), Epithelioma papulosum cyprini (EPC), Grass carp ovary (CO), and Chinook salmon embryo-214 (CHSE-214), CPE were observed. The range of the feasible replication temperature of PFRV was 15℃to 25℃. The results of study on the physico-chemical characterization of PFRV showed that the virus was completely inactivated by treatment with acid (pH 3), chloroform and heat (50℃for 30 min); however, the virus was stable to 5-iodo-2'deoxyuridine (IUdR). Ultrathin sections of cell infected with PFRV revealed the size of the typical bullet-shaped viral particle was approximately 60 nm to 70 nm in width and 105 nm to 130 nm in length. SDS-PAGE analysis of the purified PFRV indicated that PFRV genome contains 5 structural proteins, and the estimated molecular weights were 165 kDa,65 kDa, 46 kDa,38 kDa and 29 kDa, respectively.2. Cloning, sequencing and phylogenetic analysis of PFRV genome. In this study,10 pairs of degenerate primers,4 pairs of specific primers and 2 sets of rapid-amplification of cDNA ends (RACE) primers were designed.16 overlapped clones were obtained and sequenced, and the complete genome of PFRV was determined to be 11097 nucleotides in length. The PFRV genome contains five genes which are separated by the conserved sequences called'junction sequences'and are flanked with the additional sequences called'leader region'and'trailer region', respectively. In analogy to other rhabdoviruses, five open reading frames (ORFs) encoding in antigenome are named the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase protein (L), respectively, and are organized in the order 3'N-P-M-G-L 5'. The complete nucleotide sequence has been deposited in GenBank with the accession no. FJ872827. Five genes share the identical transcription initiation and transcription stop/polyadenylation signals with those of other vesiculoviruses, respectively.3'leader-and 5'trailer-sequence in PFRV show inverse complementarity, and each exhibits high homology to the respective sequences of other vesiculoviruses. Comparation of five proteins of PFRV to those of other rhabdoviruses indicates that PFRV shares higher homologies with vesiculoviruses than the rhabdoviruses belonged to other 3 animal genera, specially sharing the highest identities of 91.4%(N),55.3%(P), 76.6%(M),70.7%(G) and 87.8%(L) with SVCV. The phylogenetic trees were calculated from the deduced amino acid sequences of N, P, M, G proteins and block III within the L proteins of rhabdoviruses, the results exhibit that PFRV is mostly close to SVCV and groups with the official vesiculoviruses, and then groups with 903/87, STRV and SCRV.3. RT-PCR.2 pairs of specific primers were designed according to the conserved sequence of PFRV G gene. The concentration of Mg2+, dNTP and primers, and reaction condition including the anneal temperature and the optimal cycles were optimized. The specificity assay and the sensitivity assay were carried out according to the optimized RT-PCR condition, the results showed that only the column with PFRV served as the amplification template in gel electrophoresis exhibited the characteristic strip in detection of Infectious haematopoietic necrosis virus (IHNV), SVCV, Viral haemorrhagic septicaemia virus (VHSV), Virus nervous necrosis virus (VNNV), Infectious pancreatic necrosis virus (IPNV), Hirame rhabdovirus (HRV) and PFRV. The sensitivity in detection of PFRV was 102.5TCID50.4. Real time RT-PCR. One pair of specific primers and one probe were designed according to the conserved sequence of PFRV G gene. The concentration of primers and the probe, and the anneal temperature were optimized. The specificity assay and the sensitivity assay were carried out according to the optimized real time RT-PCR condition, the result of PFRV was positive in detection of IHNV, PFRV, SVCV, VHSV, VNNV, IPNV and HRV. The sensitivity in detection of PFRV was 101.5 TCID50.5. RT-LAMP. A set of specific primers, two outer and two inner primers, were designed based on 6 regions of PFRV G gene for RT-LAMP assay. The reaction temperature and the reaction time were optimized, and the final optimum reaction condition is keeping at 63℃for 60 min. The specificity assay and the sensitivity assay were carried out according to the optimized RT-LAMP condition, the result of PFRV was positive in detection of IHNV, PFRV, SVCV, VHSV, VNNV, IPNV and HRV. The sensitivity in detection of PFRV was 100.5TCID50,100 times more sensitive than RT-PCR,10 times more sensitive than real time RT-PCR.