An Oligonucleotide Microarray For Detection Of Capripoxviruses

Background: Poxiridae, Capripoxvirus (CPV), including Goatpox Virus (GTPV),Sheeppox Virus (SPPV) and Lumpy Skin Disease Virus (LSDV). SGP, also known assheep "smallpox", which is caused by GTPV and SPPV, is one of acute, heat, contagiousinfection sheep diseases, including Sheeppox (SP) and Goatpox (GP). The clinicalmanifestations included fever, erythema, papules, herpes, blisters (small) on parts of thehairless or less hairy skin or mucosal, visceral lesions (especially lung). SGP is one of themost serious kinds of all animal pox, as40%adult sheep mortality,100%lamb mortalityafter the onset. The disease susceptible animals and their related products are classified asstrictly restrict access to the object in countries and regions around the world, whichseriously affected international trade and sheep breeding industry. The case of GTPVinfecting human have been reported in China, Sweden and India, based on epidemiologicaland clinical symptoms, therefore, study the disease having great significance in publichealth aspect. SGPV can be used as biological weapons, which is one of the most powerfultool that the world’s terrorist threat to world peace, and is classified to Class Ⅱ dangerouswarfare agents. World Organization for Animal Health (OIE) have informed of thediseases as a statutory disease, and our country have listed them as Class Ⅰ animaldiseases. The genome of GTPV and SPPV are about150kb base-pair, a total of147ORFs.Lumpy skin disease (LSD) is caused by LSDV, and cattle to be attacked by the disease issuffering from fever, extensive nodules on skin, mucous membranes and organs surface,lymph intumescence, skin characterized by edema. The disease, also known as nodulardermatitis, infected animals can cause weight loss, a significant reduction in milkproduction, and lead to death in severe cases. LSD originated in Africa, and occurred inIsrael in1989and2006. The disease has been sho wn to escape from the African continent,and had a spreading trend.Many kinds of cattle are susceptible to LSDV, and mortality canreach3%to85%, bringing a greater loss to the economy. U.S. scientists reported LSDVgenome sequence in2001. The genome has145kb to152kb base-pair and does not havering hairpin structure. The containing of nucleotide composition is73%A+T, with aregular uniform. The homology of the three viral genes is very high, as to the gene levelfor detection of the three viruses, the specific gene sequences of the three viruses must befound. However, countries all over the world have more reliable methods for detecting theCPVs, so make the import and export inspection and quarantine in a passive position. To strictly control the introduction of such diseases and reduce losses, we must establish fast,efficient, simple and low-cost detection methods in GTPV, SPPV and LSDV as imminentas possble.Work Purpose: This project intends to study CPVs as following: Analyze the geneticcharacteristics of the viruses, and contraposing highly conserved sequences, design primersand specific probes; establish a GTPV, SPPV and LSDV oligonucleotide microarraydetection method.Research Methods: Establish the CPVs gene microarray technology, including thefollowing aspects:①Target gene sequences analysis and PCR primer design: all ORFgene sequences of GTPV, SPPV and LSDV, which published on GenBank, were comparedand found, ORF132gene relative to the others have a greater specificity, and can be atarget gene to design PCR primers;②PCR primers verification and the importantparameters optimization: Via the virus proliferation, the purpose gene cloning analysis,primer validation, the annealing temperature optimization, the number of reaction cyclesoptimization;③The microarray probes design: the connection of the PCR products,transformation, plasmid extraction and sequencing, microarry probes design and synthesis;④Preparation of microarray: using microarray micro-spotting technology, fixing sampledetection probes and all kinds of quality control probes to the chemical modificationsubstrate, amplifying the samples by antisense primer with Cy3labeled primers via PCR,then hybridization in water bath, finally, analyzing the scaned hybridization results;⑤SPPV, GTPV and LSDV microarray detection method optimization: screening specificprobes, hybridization temperature and hybridization time optimization;⑥Theverification of methodology: the validation of DNA microarray reproducibility, sensitivity,stability and other relevant parameters.Results and conclusions: The results showed that every screening probe could be with thecorresponding viral gene for specific binding, and the hybridization signal was strong andstable. This study established a specificity, sensitivity, reliability and time-saving sectionDNA microarray method, and provided appropriate technical support to animal quarantineand immigration for the future disease control. The study is to establish a set of the virusmolecular diagnostic technology platform, to meet the entry-exit animal quarantinerequirements, to prevent and control the spread of CPVs from the outside to spread toChina, and have a great significance in our country’s livestock health, safety development.