Preliminary Studies On The Production Of Transgenic Pigs Expressing Human MxA

Human MxA has been shown to exhibit a wide antiviral capacity against smallRNA viruses. This interferon-induced protein is one of the best-studied determinantsof innate immunity to viral infection. Thus, MxA has been confirmed to be a memberof the large GTPases dynamin superfamily which are involved in a variety ofintracellular transport processes. At present, the MxA-sensitive viruses includemembers of the bunyaviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses andtogaviruses, along with Hepatitis B virus.Classical swine fever （CSF） is a highly contagious disease. Controls are mostlybased on vaccination or stamping out. Although eradicated from many countries, CSFcontinues to cause serious problems in different parts of the world. In addition to theirvalue as livestock, pigs are susceptible to CSFV and can serve as reservoirs for thisvirus, allowing it to develop into an epidemic. Classical swine fever virus （CSFV） is apestivirus of the Flaviviridae family with an enveloped virion incorporatingglycosylated membrane proteins. The CSFV genome has a single strand ofpositive-sense RNA and a single open reading frame （ORF） that encodes a largeprotein that is cleaved and modified into several smaller proteins, including N^pro, C,E0, E1, E2, P7, N^S2-3, N^S4A, N^S4B, N^S5Aand N^S5B. CSFV infects pigs and replicatespredominantly in myeloid cells, causing classical swine fever （CSF）, a severe diseasethat is characterized by fever, leukopenia and hemorrhage. The development of newantiviral research for swine-raising industry has important practical significance.Mx protein has an amino-terminal skeleton, a middle domain and a carboxylterminal GTPase effector domain. When MxA protein resisting the viruses, it couldnot only inhibit viral mRNA synthesis in early transcription, but also block viralnucleocapsid into the nucleus in cytoplasm. MxA has been widely shown to inhibitthe early replication of viruses with single-stranded RNA including bunyaviruses,orthomyxoviruses, paramyxoviruses, rhabdoviruses, togaviruses, picornaviruses andASFV. However, the effect of MxA against viruses remains not entirely clear. Therearen’t any reports on whether MxA has an antiviral ability against CSFV, which do has a single-stranded positive sense RNA virus genome belonging to a pestivirus ofthe family Flaviviridae. It has been reported the expression of porcine inner Mx1wasabsent from CSFV-infected pigs because of the dsRNA-blocking effects of CSFV.Therefore, we hypothesized that the expression level of porcine inner Mx1is notsufficient to completely inhibit viral infection. For developing new ways of control ofCSF and exploring the feasibility of breeding for disease resistance to CSFV, weproduced transgenic pigs expressing high level of MxA, and did some basic antiviralresearch on the effects of resistant protection between CSFV and MxA.We constructed a vector expressing MxA in eucaryon based on PGKneotpAlox2system, the MxA cassette is under the control of Eflα promoter. After G-418selecting,40fibroblasts and70PK-15MxA-positive cell strains were obtained. We foundCSFV replicated poorly in cells stably transfected with human MxA. The proliferationof progeny virus in both PK-15cell lines and swine fetal fibroblasts （PFFs）continuously expressing MxA was shown significantly inhibited as measured by virustitration, indirect immune fluorescence assay and real-time PCR.Because of the inherently wide antiviral capacity of MxA, we aim to generate atransgenic pig model that would express a high level of MxA under the control of theEf1α promoter to protect swine against CSF. In this study, three transgenic pigsexpressing human MxA were generated by nuclear transplantation （NT）, and wedetermined the levels at which CSFV replicated in cells from different tissues of thetransgenic pigs including porcine tail tip, umbilical cord and renal endothelial cellswere isolated. We found CSFV were markedly inhibited inNo.1and No.2clone swinecells, however, the antiviral effects amone all three clone piglets were not completelyresemble. The results of CSFV-raising rate between transgenic cells and controindicated that the MxA transgenic pig has an antiviral capacity to CSFV. We presumethat the NT and reprogramming may affect the expression of MxA and other porcineinner related factors, the differences of birth weight with all three piglets proved thisopinion indirectly. Besides, the isolation and culture of virous cells were also effectedby our limitated operating-condition, this phenomenon could weak the cell vitalitydirectly.In the present study, we have a conclusion that MxA do inhibite the CSFV. We produced three MxA positive transgenic-clone swine which has the antiviral ability toCSFV. The results of this research provide a gene in transgenic breeding for diseaseresistance, provide reference data for establishment of related transgenic animals, andexpanded the anti-drug scope of the interferon downstream gene in theory.