Differentiation Of The Seven Major Lyssavirus Species By Oligonucleotide Microarray

Rabies is a worldwide zoonotic disease caused by viruses within the genus Lyssavirus, with many warm-blooded host species acting as reservoirs for infection. There are11species (7major species and4new members) and3newly discovered species which have not been classified. The7major Lyssavirus species are:species1, Classic Rabies Virus (RABV); species2, Lagos Bat Lyssavirus (LBV); species3, Mokola Virus (MOKV); species4, Duvenhage Virus (DUVV); species5, European Bat Lyssavirus Type1(EBLV-1); species6, European Bat Lyssavirus Type2(EBLV-2); Australian Bat Lyssavirus (ABLV). In addition, there are over99.8%of7major Lyssavirus species within the genus.The gold standard fluorescent antibody test (FAT) can detect the pan-Lyssaviruses, but it is unable to differentiate them. Currently the molecular methods, such as RT-PCR, RTqPCR and sequencing, were availabe to detect single Lyssavirus species in one run. However, most of them were labor-intensive, time-consuming or less-sensitive. Therefore, it is essential to establish a method for both detection and typing of the major Lyssavirus species. Oligonucleotide microarray is typically defined as a collection of microscopic spots orderly arranged onto the surface of a solid support, such as glass slide. These spots are designed so that each probe binds during hybridization reaction to a specific fluorescently labeled nucleic acid sequence corresponding to a particular gene. The level of binding between a probe and its fluorescent target is quantified by a measuring scanner. The concept of a microarray has been utilized in development of new tools for viral infection diseases detection and typing.The aim of this study is to develop an oligonucleotide microarray to differentiate all7major Lyssaviruse species, and to compare the sensitivity, specificity and accuracy with conventional methods using clinical specimens.To construct an Lyssavirus oligonucleotide microarray, the371-nucleotide region coding of the nucleoprotein gene were used to design genotypic oligonucleotide probes (oligoprobes). This region was amplified using nested RT-PCR (RT-nPCR) primer sets binding to all taxonomic Lyssavirus species. All oligoprobes were60-70nt in length which allowed a limit numbers of mismatched base pairs in the probe/target binding region, and it could be used to find novel variant species. The accuracy, sensitivity and specificity of microarray were compared with that of gold standard test FAT, conventional gel-based RT-nPCR and RT-qPCR using reference strains and clinical specimens.The main results in this study were listed as follows:Design and selection of oligoprobes. The Lyssavirus genotypic oligoprobes were design based on206N coding gene of Lyssavirus reference strains (1353nt), and E.coli plasmid pGM-T, Arabidopsis thaliana genome were used to design oligoprobes for positive and negative control, respectively. Seventy eight genotypic probes and7corresponding controls were designed with60-70nt in the range of Tm value75±5℃. After validatation by BLASTn retrieval and reference strain hybridization, finally50genotypic and7control oligoprobes were determined to construct the oligonucleotide microarray, named LyssaChip.Design of LyssaChip. Each array comprised the maximum of15×14oligoprobes in "clock-dial like" pattern. The assignment of the "clock-dial like" oligoprobes was listed as following:4located at the center and4at the vertices were green fluorescent oligomaker; each of6probes spots at12o’clock were positive (up) and negative (down) controls;12probes spots at1to2o’clock were species1(RABV);14probes spots at3o’clock were species2(LBV);17probes spots at4to5o’clock were species3(MOKV);12probes spots at6o’clock were species4(DUVV);12probes spots at7to8o’clock were species5(EBLV-1);22probes spots at9o’clock were species6(EBLV-2);14probes spots at10to11o’clock were species7(ABLV). Each control and genotypic probes were spotted duplicate, except for probe21spotted in triplicate (not mentioned in the abstract in Chinese).Construction and optimization of LyssaChip. We used commercial microarray spotting system (MicroGrid Ⅱ) for printing the "clock-dial like" oligoprobes (5’with amination group) pattern onto glass slide (treated with aldehyde group on the surface), with twelve-array block on each slide. After optimizing the labeled samples preparation, component of hybridization buffer and hybridization conditions, the standard operation procedure of LyssaChip was developed. Total RNAs were extracted using conventional methods and labeled with biotin-HEX primer by RT-nPCR, then8μl of PCR-labeled detection products were mixed with2uL of PCR-labeled positive control and10μL of optimized hybridization buffer containing2μL99%formamide,2μL50×Denhardt’s solution,2μL1×SSC,2μL2%SDS, and2μL DEPC water. Hybridization was performed at43℃for3.5h. The hybridization results were obtained by a chip scanner, and the probe with parameters of SNR=2, and F532Median-B532=800was considered as positive, otherwise was negative.Validation and evaluation of LyssaChip.We found that1to7inactivated reference Lyssavirus species can be accurately and specifically differentiated by LyssaChip. The hybridization of positive detections was only with their corresponding oligoprobes and no nonspecific reaction with unrelated oligoprobes.LyssaChip showed high sensitivity in detection of all7species. The threshold of detection of7species was2.46×105molecules/μl, except for species6which was2.46×104molecules/μl. The lowest limit of detection of species1was0.158TCID50/ml,10times more sensitive than RT-nPCR and RT-qPCR.The potential of multiplex infection by different Lyssavirus in clinical samples had been studied by mixing the species1,4,6and7templates. This indicated that LyssaChip was able to differentiate multiple viruses infection in clinical samples.To find out the best storage period and temperature, we stored6LyssaChip slides in-20℃,4℃and room temperature (20±2℃) for30days,60days,120days,150days and210days respectively. The results showed that the optimal storage conditions were-20℃at most150days.Application of LyssaChip. To evaluate the accuracy and specificity of the LyssaChip,111laboratory preserved brain specimens, including65rabies suspected brain tissues collected between2005and2010and46collected dog brain samples from Hunan province, were tested. The former were all detected as LyssaChip positive, while only64and62were positive with RT-nPCR and FAT respectively. Among46brain tissues of butcher dogs3were positive for three methods. The detection results of the three methods were summarized in Table3, which showed that the LyssaChip had100%sensitivity (CI94.48%-100%) and93.94%specificity (CI82.10%-98.63%) compared with FAT. The LyssaChip had very high consistency with FAT (κ=0.944) and RT-nPCR (κ=0.981).LyssaChip was further used in2010international Laboratory Test for Rabies (ILT-2010), a set of12samples (one each of RABV, EBLV-1, EBLV-2and ABLV as positive controls,1negative control, and7blinded samples) received from the European Union Reference Laboratory for Rabies (EU-RL) at the French Agency for Food, Environmental and Occupational Health Safety (ANSES). The samples were also tested by FAT, RT-nPCR and RT-qPCR at the same time. Results revealed that the4methods were100%consistent, but the LyssaChip was additionally able to differentiate between the species within8h, with all12samples tested on a single chip slide, while the FAT and RT-nPCR could not differentiate them.The LyssaChip developed in current study represents a rapid, high-throughput and economic method for the detection and differentiation of7traditional Lyssavirus species. The entire detection procedure takes about8h, including1h for RNA extraction,3h for RT-nPCR labeling,3.5h for hybridization and30min for washing and scanning. Conventional RT-nPCR and sequencing methods are unable to accomplish the same task within such short time. Additionally, the LyssaChip can assay12samples on a single slide, and permitted the simultaneous processing of at least7-8slides at a reasonable cost (approximately35RMB per sample).In conclusion, given its high sensitivity, specificity, speed and low cost, the LyssaChip is a potential tool for clinical laboratories for detection and differentiation of7traditional Lyssaviruses.