Research And Application Of Oligonucleotide Microarrary For Classical Swine Fever Virus And Porcine Reproductive And Respiratory Syndrome Virus

Classical swine fever (CSF) is a highly contagious disease leading to significant economic losses for pig industry of our country and the world. The causative agent, classical swine fever virus (CSFV) is a Pestivirus of the family Flaviviridae. Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV). In 1990's, PRRS spread to China. In China in 2006, the highly pathogenic PRRS outbroke in large scale, great damages have took place in pig industry. The PRRSV Nsp2 mutant strains were confirmed one of significant etiological agent for the highly pathogenic PRRS. CSF and PRRS have become the most important swine diseases for pig industry in China. Therefore, it is of great importance to develop robust diagnostic methods for the prevention and control of them. DNA microarray is solid-phase hybridization based on the principle of nucleic acid hybridization between attached cDNA or oligonucleotide and the target made from the samples, by the detection and analysis for hybridization signal, the sequence information of the sample or the gene expression profile will be obtained.The purpose of this study is to develop a microarray method for differential diagnosis of the highly pathogenic PRRSV Nsp2 mutant strains and classical Amercian PRRSV and to develop a CSFV genotyping microarray. The principle of the study for the PRRSV microarray is to design American PRRSV-common probes and probes for differentiation of the highly pathogenic PRRSV Nsp2 mutant strains. By the hybridization of targets and the probes attached on the slide, all the American PRRSV strains can be detected and the Nsp2 mutant strains can be distinguished. The principle of the study for CSFV genotyping microarray is to design and select specific probes to each of ten subgroups within the three gene groups according to partial E2 gene of CSFV. By the hybridization of targets and genotyping probes, the subgroup of the tested CSFV strains can be determined.The main results of this study are as following:1 Development and application of oligonucleotide microarray for PRRSVThe nucleotide sequences of American PRRSV and the highly pathogenic PRRSV Nsp2 mutant strains were retrieved from NCBI GenBank. On the basis of alignment of sequences, one set of duplex PCR primers were designed to amplify the conservative region of American PRRSV genome and partial Nsp2 gene sequence covering the two non-continued deletions. The 31～35 mer oligonuleotide probes were designed to bind the multiple target sites within the conservative region of American PRRSV genome and the sequence corresponding to the deletions of Nsp2 gene.5 Amercian PRRSV-common probes and 9 highly pathogenic PRRSV differentiation probes were designed. The Tm value of probes is between 85.0℃and 90.9℃. In addition,4 positive control probes were designed corresponding to the sequence of enhanced green fluorescent protein gene.1 monitor probe was designed according to sequence of a plant gene, which was used to address the probes in the microarray and monitor the result of spotting.1 random 35mer sequence was synthesized as negative control.20 oligonuleotide probes in total were designed in this study.To prepare microarray, different probe concentrations for spotting, spotting buffer and the immobilization time of probe were tested and optimized. By using American PRRSV and the highly pathogenic PRRSV cDNA as PCR template respectively, Cy3 labeled DNA targets were successfully amplified in the duplex PCR for amplification of the conservative region of American PRRSV genome and partial Nsp2 gene fragment covering the two non-continued deletions.Experiments were carried out to optimize the most optimum hybridization buffer and the concentration of formamide in hybridization buffer. In these experiments, the specificity of the oligonucleotide probe was tested.4 American PRRSV-common probes and 4 probes for highly pathogenic PRRSV differentiation which had good specificity were selected to fabricate the microarray at last.The sensitivity and specificity of the microarray was tested. The results showed that the sensitivity of this assay was equal to the routine RT-PCR method for differentiation of the highly pathogenic PRRSV. The oligonucleotide probes did not show cross reaction with samples of CSFV, Porcine circovirus 2 (PCV2) and Pseudorabies Virus (PRV).12 clinical PRRS suspected samples were tested by this assay,1 case was identified as classical American PRRSV nucleic acid positive,5 cases were Nsp2 mutant strains nucleic acid positive,6 cases were PRRSV nucleic acid negative. At the same time, the samples were tested by the routine RT-PCR method for differentiation of the highly pathogenic PRRSV. The results of RT-PCR was the same to the oligonucleotide microarray. The result indicated that the oligonucleotide array could be used as an potential tool for differential diagnosis of the highly pathogenic PRRS and classical American PRRS. Because the sensitive RT-PCR method may lead to nonspecific amplification sometimes, in this study, several probes were designed according to the two PCR fragments, by hybridization, it can rule out false positive result brought by RT-PCR. Furthermore, more microarray can be spotted on the same slide, so this method is more suit to the parallel detection and differential diagnosis of samples in bulk for epidemiological investigation.2 Development and application of oligonucleotide microarray for CSFV genotypingIn this study 210 CSFV viral sequences belonging to 3 gene groups and 10 subgroups were collected, comprised of complete genome, complete E2 gene sequence and 190nt partial E2 gene sequence coding major antigen site. The compiled sequences were aligned within the 190nt sequence by clustalxl.81 software. Corresponding to the conserved sequence of each subgroup,29～37 mer oligonucleotide probes to different subgroups were designed by Oligo 6.0 software. To ensure the specificity of the probe for its target, the number of mismatched bases between the probe and it's target subgroup was no more 3, while between the probe and the other subgroups, it was no less 3.86 oligonuleotide probes in total were designed in this study.Deploying the CSFV specific nested RT-PCR method recommended by the EU diagnosis manual for CSF, using the outer set PCR product as template, Cy3-dCTP was incorporated during inner set PCR. In this way, CSFV subgroup 1.1, subgroup 2.1, subgroup 2.2, subgroup 2.3 targets were amplified and labeled. For subgroup 1.2, subgroup 1.3, subgroup 3.1, subgroup 3.2, subgroup 3.3 and subgroup 3.4, targets were amplified using synthesized E2 gene fragment as template by inner set PCR primer pair.Different hybridization buffer was tried and the concentration of formamide was optimized for the specific hybridization. The specificity of the oligonucleotide probe was tested and the probes which cross reacted with PCR targets non-specifically were deleted.41 probes which had good specificity were selected from the total 86 ones at last. The selected probes were 5 probes for subgroup 1.1,6 probes for subgroup 1.2,2 probes for subgroup 1.3,4 probes for subgroup 2.1,4 probes for subgroup 2.2,2 probes for subgroup 2.3,5 probes for subgroup 3.1,4 probes for subgroup 3.2,5 probes for subgroup 3.3,4 probes for subgroup 3.4.By hybridization of representative samples of various CSFV subgroups with microarray, according to the Signal to Noise Ratio (SNR 532) of probes, SNR≥1.6 was defined as the cut-off. Although weak cross reaction was found when targets of subgroup2.2, subgroup2.3, subgroup3.1 and subgroup 3.3 hybridized with the microarray, the SNR of non-specific probes was closed to the cut-off and far below that of specific probes, by hybridization pattern or SNR, the result could be determined.The sensitivity and specificity of the method was assessed. The results showed the sensitivity of this assay was the same to the published CSFV Real-time RT-PCR method developed by our research group. The experiment for specificity showed that the oligonucleotide probes did not hybridize with samples of PRRSV, PCV2 and PRV.For lack of samples of CSFV subgroupl.2, subgroupl.3 and gene group 3,16 CSF positive samples representing subgroup 1.1, subgroup2.1, subgroup2.2 and subgroup2.3 were tested to validate the CSFV microarray. The result showed that the subgroups of all the samples could be identified accurately by hybridization pattern.The high-throughput and differential diagnostic method is important to epidemiologic study and the diagnosis of disease. Hitherto, reports on the study of microarray for CSFV genotyping and differential diagnosis has not been found. The results of this study showed the promising application of the microarray for differential diagnosis of the highly pathogenic PRRS and classical PRRS and genotyping of CSFV. Although a rapid increase of research on DNA microarray for pathogen detection has been witnessed, most of the technologies are not applied to the routine detection. It is believed DNA microarray will be widely used as routine diagnostic method with the further developments of technology and equipments.