Construction And Identification Of Immortal Ovis Aries Cells Line Supporting Stable Proliferation Of PPRV

Peste des petits ruminants virus(PPRV),a critical member of genus Morbillivirus in the family Paramyxoviridae,can cause a highly Contagious and infectious diseases in both wild and domestic small ruminants with clinical features including fever,pneumonia,diarrhea and inflammation of respiratory tract mucosa.PPRV is an enveloped virus,and its genome is a single-strand,negative-sense RNA molecular,which contains 15948 nucleotides encoding 8 proteins(N,P,C,V,M,F,HN and L).It's well known that cells line are important platforms for studying on the pathogenic mechanism of viruses or developing vaccines against viral disease.However,many visuses(like RHDV and PPRV)lack continuous cells line supporting their stable proliferation in vitro,and this situation greatly restricted the foundational and applicative research of these viruses.To overcome the problem,some immortal technologies of primary cells are invented and widely used.In this study,we aimed to construct an immortal sheep cell line supporting stable proliferation of PPRV.Firstly,we constructed a recombinant plasmid pLOV-puro-hTERT.Then primary sheep lung fibroblast cells were infected with the recombinant lenti-virus rescued from 293-T cells.After 48 h post infection,the positive cells were screened with puromycin.After screened about 10 passages,the immortal sheep lung fibroblast cells were got and named SLT cells line.Unfortunally,though the results of RT-PCR and Western blot(WB)showed that SLT cells line coud support PPRV proliferation,the typical CEP of PPRV infection couldn't be observed,and PPRV couldn't be detected in SLT cells after passaged 5 times,that meaned that the SLT cell line can't support PPRV satble proliferation.It is reported that goat signaling lymphocyte(SLAM)Activation Molecule is an important cell receptor acquired for PPRV binding to target cells.Therefore,we further constructed another cell line stably expressing gSLAM protein utilizing lenti-virus system and the designed cells was named SLT-gSLAM cells.Finally,SLT-gSLAM cells were infected with PPRV Nigeria /75 strain,and typical CPEs were observed after passaged 3 times,and the recovered viruses could passaged more than 10 times continuously.The results of RT-PCR and WB all showed that PPRV could proliferate in the SLT-gSLAM cells line and the viral titer can reach 105.6 at 48 h post infection.In a word,we have successfully established a sheep cell line supporting PPRV stable proliferation in vitro,which would provided an excellent platform for studying on the molecular pathogenic mechanism of PPRV.