Construction And Antitumor Efficacy Of RClone30 Chimeric Virus Expressing The HN Gene Of Mesogenic Strain Anhinga

Newcastle disease virus(NDV), which is a single-stranded, negative-sense, non-segmented RNA virus, belongs to paramyxoviridae and avian paramyxovirus genus. In the 1964, NDV was firstly discovered for cancer therapy and become one of the hot spots in the field of cancer research. According to the pathogenicity to chicken, NDV can be divided into velogenic strain, mesogenic strain and lentogenic strain. The oncolytic effect of velogenic strain is good, but it is a threat to the ecological and affects the development of the poultry industry; the lentogenic s train can not affect the development of poultry, but the oncolytic effect of the lentogenic is poor. Choosing what kind of strain as agent to treat tumors is always major issue to make a decision in this research field. Previous studies showed that NDV F g ene is the key gene of NDV oncolytic effect, but the oncolytic effect of F gene is related to the virulence. A number of researches show that the replacement of F gene leads to a remarkable enhancement in oncolytic activity accompanying with the increase in virulence. So F gene is not an optimal candidate to improve the oncolytic activity of NDV.Recently, it is reported that the mutation in the HN gene significantly improved the ability of syncytium formation of the virus by the Qingzhong Yu team in South eastern United States Institute of poultry. In addition to the F gene, HN gene may also be a n important factor to decide the viral oncolytic effect. Meanwhile, NDV HN protein is a multifunctional protein and plays an important role in the viral infection process. HN protein has two different bioactivities, hemagglutinin and neuraminidase, which can recognize the sialic acid receptors on cell surface and improve the fusion activity of F protein. Although the role of HN gene in the process of virus infection has been deeply studied, at present, the exact relationship of HN gene and NDV oncolytic activity was still unknown. In this study, we replaced the HN gene from lentogenic Clone30 with the one from mesogenic strain Anhinga. The chimeric virus r Clone30-Anh(HN) will help us to understand the relationship between HN gene and oncolytic activity of NDV. It is provide the theoretical foundation to development of safe and effective oncolytic agent, namely high oncolytic effect and maintaining the safety of lentogenic strain. To substitute the HN gene on the DNA level, we firstly cloned the full length genome of Clone30 strain by reverse genetic manipulation technique. A piecewise cloning method was adopted to construct a full-length c DNA clone of NDV strain Clone30, ten overlapping sub-genomic c DNA clones spanning the entire NDV genome were generated and sequentially cloned into a low-copy-number vector designated PFLC. The sub-genomic cDNA fragments were joined by over-laps taking advantage of unique restriction enzyme sites naturally present in the virus genome. Full-length c DNA clone was modi?ed by site-directed mutagenesis at position 3520 bp, 6178 bp and 12361 bp of the virus genome. These sites were served as molecular tag to differentiate the rescued virus from the parental one. In this study, the generation of infectious NDV entirely came from cloned c DNA. This system made use of BHK-21 cell line, stably expressing the T7 RNA polymerase. The rescued virus was first inoculated into embryo eggs and the allantoic fluid was harve sted for the late experiments, the rescued viruses were named r Clone30. In this study, we replaced the HN gene from lentogenic Clone30 with the one from mesogenic strain Anhinga. The chimeric virus r Clone30-Anh(HN) will help us to understand the relationship between HN gene and oncolytic activity of NDV. First, we inserted the PCR production of HN gene into the p Clone30 vector, then co-transfected the vector with assistant plasmids(NP, P and L) into BHK-21 cells. After incubation at 37°C for 72 h, cells were frozen and thawed in-80°C for three times. 9-11 day SPF chicken embryos were inoculated with the above cell supernatant and maintained at 37 °C. Allantoic fluid which containing the chimeric virus r Clone30-Anh(HN) were harvested after 72 h.One-step growth curve experiment showed that the chimeric virus had a same growth characteristic with the parental strain. The replacement of HN gene did not affect the replication of r Clone30. The HA titer of chimeric virus(24) was similar with Anhinga strain(24) but lower than parental strain Clone30(29). Confocal microscope data showed that the chimeric virus was more efficient in inducing cell fusion compared with parental strain, which was detected as the bigger syncytia in Hep G2 cell monolayers infected by chimeric virus. The virulent and pathogenic analysis of chimeric virus rClone30-Anh(HN) revealed that there were no significant different between rClone30-Anh(HN) and parental strain(TCID50=3.95×107),(MDT>120h),(EID50=1.8×108). The ICPI of chimeric virus(ICPI=0-0.5) was slightly higher than parental strain(ICPI=0), but the pathogenicity still belongs to lentogenic virus. The result demonstrated that replacement of HN gene did not make an obvious affect in virulence and pathogenicity on Clone30 strain. The MTT results demonstrated that rClone30-Anh(HN) performed a higher cytotoxicity to Hep G2 cells than parental strain. The capacity of two viruses to induce tumor cells apoptosis was detected by Annexin V-PI assay, Rhodamine 123 staining, and Caspase3 transcription level. All the results proved that the replacement of HN gene improved the capability of r Clone30 to induce tumor cell apoptosis. In vivo, ICR mice carrying tumor of hepatoma cells H22 were treated via intratumoral injection of r Clone30-Anh(HN) and rClone30. The treatment of chimera showed an obvious suppression in tumor volume. The pathological section demonstrated that the chimeric virus group had a severe T cell infiltration and necrosis. Animal experiment data proved that replacement of HN enhanced the tumor suppress effect of Clone 30 strain.In conclusion, the constructed r Clone30-Anh(HN) carrying the HN protein of mesogenic Anhinga strain showed similar characteristics of virulence and pathogenicity and enhanced tum or suppress capability compared with parental strain. These results provide a new approach to development of good oncolytic effect, low pathogenicity oncolytic virus. This study revealed that HN gene plays an important role in NDV oncolytic process. The chimeric virus rClone30-Anh(HN) may become a new candidate for tumor therapy.