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G.Nagarajana*, Shelesh Kumar Swamia, Shyam Singh Dahiyaa, S.D.Narnawarea, S.C.Mehtaa, P.K.Singha, Raghvendar Singh , F.C.Tutejaa and N.V.Patila
a National Research Centre on Camel, Post Bag No.7, Jorbeer, Bikaner, Rajasthan-334 001,  India
*Corresponding author
Dr. G. Nagarajan, Scientist, National Research Centre on Camel, Post Bag No. 7, Jorbeer, Bikaner � 334 001, Rajasthan, India
Email:  HYPERLINK "mailto:camelnag@yahoo.com" camelnag@yahoo.com
Phone: +91-151-2230183; Fax: +91-151-2231213
Key words: Camel, Contagious ecthyma, Pseudocowpoxvirus, UDG, India

















Calf mortality is  the most important health problem faced by camel (Camelus dromedarius) herders, even sometimes reaching 50%, It is important to examine any potential factor, which may contribute to the debility of camel calves and hence to such mortality (Richard, 1979).
The disease Camel Contagious Ecthyma (CCE) is widely recognized in camel-rearing regions of the world (Housawi et al.,2004). Recently, pseudocowpoxvirus (PCPV) has been reported as the etiological agent of CCE (Abubakr et al.,2007; Nagarajan et al.,  2010). The effect of CCE was not only due to mortality of camels but also to loss of weight, poor growth rate, decreased milk yield and also the cost of medicines.(Khalafalla ,1998).
Several orthopoxvirus (OPV) enzymes are being considered as targets for antiviral drugs and  the important genes of PCPV from the camels have yet to be characterized. As the UDG protein is highly conserved between vaccinia and variola major virus, an inhibitor could be helpful to decrease the pathogenicity to all orthopoxvirus orthologs (De Silva and Moss, 2003).
In the line of the development of poxviral inhibitor, the present study was taken up as a preliminary step to understand the sequence homology of the UDG encoding gene of PPV from the camel with rest of the Poxvirus.
During the epidemiological survey conducted in the mid August 2011 at various camel inhaitating areas of Rajasthan, India, it was observed that camel calves of below one year of age of either sex in a camel herd (four males and six females) at Jagthi village of Udaipur district, Rajasthan state, India were showing the exanthematous skin lesions around the facial region and were suspected to be infected with contagious ecthyma. .Scab materials were collected from three severely affected animals and stored at -200C.
Total genomic DNA was extracted from collected skin scabs using AxyPrep Multisource Genomic DNA Miniprep kit (Genaxy Scientific Pvt. Ltd.) according to the manufacturer�s instructions. As per our previous report (Nagarajan et al., 2010), the nucleotide sequences of the envelope gene amplified from PPV DNA of camel skin scabs suspected for contagious ecthyma is found to be closely related to PCPV. Therefore, in the present study, nucleotide primers were designed using the coding sequences of  uracil DNA glycosylase (UDG) encoding gene of PCPV isolate from Finnish reindeer (GenBank accession No.GQ329669); The primer pairs for UDG encoding gene , forward   (UDGF) 5�actgggatccatggcgacgccggcggccggcgcgctgcg 3� , reverse (UDGR) 5�gtacgaattctta attttcactataaaaagaaaatcctcg3�. Polymerase chain reaction (PCR) amplification of the UDG encoding gene was performed using the following thermal profiles: initial denaturation at 940C for 5 min, followed by 35 cycles of denaturation at 940C for 1 min, annealing at 570C for 1 min, extension at 720C for 1 min, and final extension at 720C for 10 min. The PCR-amplified products were checked by electrophoresis on a 1% agarose gel. After purification of the amplified products from the low melting point agarose gel by phenol extraction followed by ethanol precipitation, the fragments were cloned into pGEM-T Easy Vector (Promega). The ligated mixtures were then used for transformation into Escherichia coli DH 5� (Sambrook et al., 1989). Positive clones were identified by colony PCR using gene- specific primers and restriction analysis with EcoRI. 
Sequencing of the PCR amplified DNA fragment (three from each geographical area) in both directions was carried out in an automated DNA sequencer at sequencing facility of Delhi University (South Campus), Delhi. Sequences of the virus isolates from Bikaner, Pali and Udaipur were submitted to NCBI GenBank and assigned the accession numbers JQ728422. Nucleotide identity, amino acid identity and comparison of the sequences with published sequences of members of PPVs available in the GenBank database were carried out using the computer software BioEdit version 7.0.9. These sequences were compared in Clustal X (Thompson et al., 1997) and a phylogenetic tree was constructed based on the amino acid sequences by the neighbor-joining method using Mega 5 (Molecular Evolutionary genetics Analysis software with bootstrap values calculated for 1,000 replicate (Tamura et al., 2011). The open reading frame (ORF) and translation of nucleotide sequences to amino acid sequences were predicted by using the computer software Generunner version 3.05 (hastings Software inc. Hastings, NY, USA; HYPERLINK "http://www.generunner.net"http://www.generunner.net).
The complete nucleotide sequences of UDG encoding gene of camel PCPV from Udaipur, India was compared to corresponding amino acid sequences from two OPVs and seven other parapoxvirus (PPV) sequences available in the NCBI database (Fig.1). The open reading frame (ORF) of UDG encoding gene of camel PCPV is 696 bp encoding a polypeptide of 26.0 kDa. `
Sequence analysis revealed that UDG encoding gene of camel PCPV from Udaipur shared 95.8 - 96.9 % and 95.2 � 96.1 % sequence identity at the nucleotide and amino acid level, respectively with orf viruses (ORFV) isolates from different geographical areas of the world. Further camel PCPV exhibited 93.1 % and 91.7 % sequence identity at nucleotide level with human PCPV and reindeer PCPV strains. But, at the amino acid level, both human and reindeer reference strains of PCPV had equal identity with camel PCPV i.e., 91.8%. Bovine popular stomatitis virus (BPSV) reference strain exhibited 81.1 % and 78.5 % identity with camel PCPV. With two OPVs considered in the study, PCPV camel covered 52.4% homology at nucleotide level whereas 55.4% and 55.8% amino acid homology with CMLV and VACV, respectively (Table 1). 
All the three active-site residues (Tyr-70, Phe-79, and Asn-120) of vaccinia virus involved in the formation of DNA recognition pocket are retained in all the eight PPV isolates and two OPVs considered in the analysis of the present study. Similarly two amino acid residues (Asp-68 and His-181) of VACV-UDG protein involved in the cleavage of glycosylic bond are retained in all the ten poxvirus species considered in the present study (De Silva and Moss, 2003)(Fig.1).
Analysis of phylogenetic tree constructed using amino acid sequences of the UDG encoding gene revealed that camel PCPV from Udaipur clustered with ORFV isolates from different geographical areas of the world (group I), published earlier, supported by high bootstrap values. But, reindeer PCPV and human reference strain PCPV (group III) clustered together forming another group (group II) while group III is formed by CMLV and VACV isolates (Fig.2). 
Based on the findings of our previous study, the IL-10 encoding gene  of camel PPV from Udaipur, clustered with PCPV (Nagarajan et al., 2013) but our present finding highlights that the UDG encoding gene of  camel PPV (from the same place and year of collection) clustered with ORFV.
 Considering the findings of our earlier and present study, it is assumed that the possibility of incorporating different genes from different PPVs by camel PCPV cannot be ruled out. This assumption can only be confirmed and so we suggest the full length sequencing of camel PCPV genome.
Acknowledgements
The authors are thankful to Dr. P.N. Sivalingam, Scientist, CIAH, Bikaner, India, for the analysis of the sequence data. The help rendered by M.L. Kiradoo, Lab Attendant, NRC on Camel, Bikaner and Manoj in the collection of biological samples from the camels is also gratefully acknowledged.
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