Studies On Pathogenisis And Genomic Analysis Of Orf Virus Isolates In Fujian Province

Contagious ecthyma,also known as Orf,is caused by the Orf virus(ORFV)and is a contagious skin disease of goats,sheep,humans and other animals.Clinically,the disease is characterized by the formation of papules,vesicles,pustules and scabs onthe skin,and in the mucosa of the lips,noses,tongues and breasts.In recent years,goat raising has developed rapidly.There have been occasional reports of cases of orf,but they lack information regarding pathogen isolation and identification and ORPV biological characteristics.It is therefore imperative to illuminate the current epidemiological status of Orf in the Fujian province and to develop a vaccine against ORFV infection.The main results of our research were detailed as:1.First,scab samples were collected from suspected ORFV infected goat herds in several regions of Fujian Province(China)from 2011 to 2012.The 8 novel ORFV isolates were successfully isolated by use of primary ovine fetal turbinate(OFTu cell),and identified as ORPV by PCR amplification of the ORFV011 and ORFV059 genes.The 8 isolates were named as OV?GO,OV?SL,OV-YX,OV-SJ1,OV-NP,OV-FQ,OV-DS and OV-GT,respectively.The OV?YX isolate was further identified by transmission electron microscopy,pathologic histology and immunohistochemistry.Pathological findings in this case revealed excessive hyperplasia of skin keratosis,ballooning degeneration in keratinocytes and inflammatory cell infiltration.Irnmunohistochemistry results revealed that the virus antigen was localized in the spinous cells in the stratum spinosum.Tissue culture infective dose(TCID50)of four ORFV isolates of the cell cultures were detemined,and the TCID50 of OV-YX P3,oV-GO P4,oV-SJ1 P3,oV?NP P4 were 10-6.5/0.1 mL,10-7.6/0.1 mL,10-6.1/0.1mL and 10.6 7/0.1mL,respectively.2.In this study,phylogenetic trees based on the ORFV0ll and ORFV059 genes of 11 Fujian ORFV isolates and other isolates from GenBank were constructed to determine the relationship of ORPV isolates from Fujian province with other isolates from China and abroad.We found high heterogeneity between isolates from 10 farms in Fujian Province.Only two isolates from locations within 1 km to each other had the same viral genes.There is no correlation between the geographical distance between the corresponding collection sites and the phylogenetic distance in ORFV011,ORV059 or ORV011-059 sequences for any pairing of two isolates.The genetic variety was mainly due to interspecies difference.Anti-ORFV serum antibodies were detected by indirect ELISA for 349 serum samples collected from 15 goat farms in four regions of Fujian Province,and the results showed that the positive rate was 64.47%,indicating that ORFV infection was common in the goat herds in Fujian Province.Western blot results showed that a 40 kDa protein was the dominant antigen in ORFV infection.3.The morphogenesis of orf virus(ORFV)virulent strain YX in OFTu cells and the ultrastructural changes of host cells were observed and analyzed using transmission electron microscopic techniques.This approach was effective,and we determined that the best observation time was within 24 h of infection.Results revealed that the endoplasmic reticulum,mitochondria,golgi apparatus and the nucleus showed greater ultrastructure changes in ORFV infected cells.At 12 h post-infection,immature virus particles were first observed in the endoplasmic reticulum.At 18 h post-infection,virus particles in different developmental stages were observed,and the envelop of virions was presumably derived from the golgi network.Once the virus particles matured,they immediately moved towards the cell membrane,releasing virus particles through budding.4.Based on phylogenetic trees of ORFV01 1 and ORfV059 nucleotide sequences and the geographical distribution,4 typical ORFV isolates(OV-GO,OV-YX,OV-NP and OV-SJ1)were sequenced by next generation sequencing to determine the whole genome sequence for analysis of the genetic diversity at the genome level.The four isolates ere 132 to 139 kb in length,each containing 124 to 132 genes and about 64%G+C content.Great variation appeared in different isolates,and the most notable differences between the four isolates were found near the genome termini.Genes at the end of the genome shared low identity among different isolates.In the GO,YX,and SJ1 genomes,one predicted gene appeared twice completely located within the inverted terminal repeat(001/134).However,in the NP genome,only a partial sequence of one predicted gene was located in the inverted terminal repeat.In the NP genome,gene 001 is intact,but the corresponding 134 gene was disrupted due to the lack of initiation coding.By comparison of whole genome sequences,it was surprising to find no sequence corresponding to genes 114 to 120 genes(7 genes in total,about 5.5 kb)in the OV-NP genome.Genes 117,118 and 119 were deleted in OV-SJ1 genome(3 genes in total,about 1.5 kb).The phylogenetic trees based on the deduced amino acid sequences of each ORFV gene from this study and GenBank were constructed by Neighbor-joining Algorithm Method using MEGA 5.0 software.Among 132 genes of ORFV,16 genes showed great variations between goat ORFVs and sheep ORFVs with gene 008 showing the greatest variation.This study provided the first complete sequence of goat ORFV isolates from China,which helped us to understand the genetic diversity of ORFV in the region.5.An animal experiment was set up to investigate the virulence of OV-YX,OV?SJI,OV-GO and OV-NP.Clinical symptoms and histopathology indicated differential virulence of OV-GO,OV-YX,OV-SJ1 and OV-NP,in descending order.OV-NP and OV-SJ1 with several gene deletions showed low virulence while OV-GO and OV-YX without gene deletions showed strong virulence,indicating gene deletion resulted in an attenuated disease phenotype in the natural host.The 2 attenuated ORPV isolates may lay the foundation for the further research of an ORFV vaccine.