Complete Genome Sequence Analysis Of Goatpox Virus And The Regulatory Mechanism Of Host Antiviral Innate Immunity

Goat pox,which is caused by Goatpox virus(GTPV),is an acute,pyrexia,highly contagious endemic disease and is one of the important infection in the goat industry.Clinically,the disease is characterized by a rise in temperature(40?42?),nasal and ocular discharge.Round or oval papule or small pox appearing on the whole body skins and mucosae of respiratory and digestive tract.It is list as one of the diseases which must be reported to World Organisation for Animal Health(OIE)and is classified as the 1st animal disease in China.In order to understand molecular characteristic of GTPV and antiviral innate immune of host and to further develop new vaccines and therapeutic agents against goatpox,one GTPV strain was collected from a goat flock in the suburbs of Fuzhou in October 2012 and researches on the etiology,pathology and pathogenesis of GTPV were performed.The autopsy of the sick goat results showed that gray pox in different sizes were observed on the whole body skins,and on the surfaces of livers,lungs and the serosal surface of rumens.Histopathology showed obvious skin keratinizing,inflammatory cells increased,the acidophilic inclusion bodies in cytoplasm of prickle cells,interstitial pneumonia,epithelioid cells of lung alveoli proliferation,fatty and vacuolar degeneration in the liver,splenic nodules shrink or disappearing,transudation of renal capsule,hemolysis phenomenon in the renal tubular and a degenerative changes in the epithelial cells of tubules with proteinuria and hemolysis.Homogenates from tissues collected from the outbreak were inoculated into Ovine fetal turbinate(OFTu)cells to isolate virus.The cytopathic effect(CPE)was observed after second blind passage and CPE appeared regularly 48 hpi after the third passage,manifesting cell rounding,clustering,diopter increasing,cell detachment and cavity.Virus was successfully isolated and named as GTPV_FZ.Mass-culture of virus was purified by discontinuous sucrose gradient.Two major virus bands were observed in the 36%?40%and 24%?28%sucrose gradient,but the most abundant and intact virions were seen in the band of 36%?40%.By scanning electron microscopy(SEM),virions were round,ovoid,or bricks,with rough surface,like a straw hat shape around the middle uplift,sag of virus particles,the size with about 250 nm to 350 nm.The electron microscope examination showed two types of virus particles.One was virus particle with transparency in the centre and the other virus particles was condensed,all with the envelops.The morphogenesis of GTPV in OFTu cells and the ultrastructural changes of infected cells were observed and analyzed by using transmission electron microscopic technique.The virions were enveloped,round or ovoid in shape with diameter of 250?350 nm.Dumbbell shape DNA core and two corpuscles were observed in some virions.Viruses replicate and breed in the cytoplasm.The mature viruses were released from infected cells by budding or cell broken.The main ultrastructural changes of the infected cells were as follows:increased number of cytoplasmic vacuoles;expanding endoplasmic reticulum like a capsule full of floccules with pool separation,the characteristic morphological changes of apoptosis,mitochondria showing hyperplasia,swell,dark and vague,Golgi budding.Finally,infected cells were destroyed by viruses and dissolving.Residual virus inclusion body were observed in some infected cells.The genome of GTPV_FZ was sequenced by Illumina genome analyzer platform.Contigs were assembled into a single genomic sequence of 150,194 bp,containing approximately 25%G+C content uniformly distributed within the genome.GTPV_FZ contains 151 putative genes of 42 to 2,008 amino acids in length.Terminal hairpin loops in the genome contain two identical inverted terminal repeat(ITR)regions of 2301 bp.Two putative genes each appear twice because they are completely located within ITRs(GTPV001,002,146,151).(1)The complete genomic sequence analysis shows high variation in GTPV_FZ,especially in the terminal regions showing the highest frequency of genetic differences.Comparison of GTPV_FZ with GTPV_PL and GTPV_GV showed high variation containing 765 and 774 bp changes,respectively,and 3 genes are less than 90%identical(GTPV007,072,129.5)in amino acid identity.(2)GTPV_FZ shares an average of 96.9%nucleotide identity when comparing to the complete genomic sequences of the SPPV strains.In amino acid identity,GTPV_FZ and SPPV_SA share 4,96,44 and 5 ORFs with 100.0%,96.0%?99.9%,90.0%?95.9%,80.0%?89.9%identity,respectively.(3)The complete genome nucleotide identities of GTPV_FZ to LSDV strains NI-2490,NW-LW and LW-1959 are 97.4%,97.8%and 97.8%,respectively.(4)Phylogenetic analysis based on the complete genome showed that the nine strains clustered into three branches,with each species of CaPVs,GTPV,SPPV and LSDV,forming a separate branch.Phylogenic tree based on complete genomes also showed that GTPVs were more closely related,genetically,to LSDVs than to SPPVs.To better understand the pathogenesis of GTPV in goats,we performed experimental infection of goats and cells with the viruses.Our results showed that GTPV infection doesn't activate host innate immune signaling in OFTu and GSF cell,while GTPV infection in goats activated the Toll-like receptors 2/9(TLR2/9)signaling pathway to induce the nuclear translocation of interferon regulatory factors 3/4(IRF3/4)that resulted in the expression of type I interferon(IFN)(IFN-?,IFN-?),inflammatory cytokines including interleukins(IL)(IL-1?,IL-6,and IL-18)and tumor necrosis factor-?(TNF-?),some critical interferon-stimulated genes such as interferon-induced transmembrane protein 3(IFITM3)and interferon stimulated gene 15/20(ISG15/20).Of note,in the present study,we observed that GTPV infection induced a high expression of SOCS-1,p-STAT3 and proinflammatory cytokines in the spleen and lung.These results indicated that GTPV infection activated host innate immune signaling and thereby triggers available antiviral innate immune response in vivo.In this study,the purified viral proteins of GTPV_FZ used as antigen to immunize BALB/c mice.The spleen cells were fused with Sp2/0 myeloma cells by a conventional method.Mabs was screened by indirect ELISA method and indirect immunofluorescent assay and cloned by limited dilution method.A total of 2 hybidoma cell lines were developed,which could steadily secrete monoclonal antibody with IgM subtypes against GTPV were obtained.The prepared McABs to GTPV can be used for rapid diagnosis and provide a useful tool to further study pathogenic mechanism of GTPV.