Parallel Screening Of Medical RNA Viruses On Genus Level By Microarray Technique

More than 70%of human infectious diseases are caused by viruses,which are termed as viral infectious disease.Among 5400 known viruses,about 400 viruses are related with human diseases.And more than 80%of human pathogenic viruses are RNA viruses.Most of the high pathogenic viral diseases are infected by RNA viruses,such as Ebola and Marburg virus in Filovirus,Lassa virus in Arenavirus and Venezuelan equine encephalitis virus in Alphavirus.It is extremely important to establish methods for high throughput and parallel screening and identification of unknown RNA viruses. Microarray(genechip) is a large scale integrated solid-phase hybridization technique.Its fundamental principle is the hybridization of nucleic acid molecules.Based on the principle of complementarily match of double-strand DNA,oligonucleotides,cDNA or gene fragments with known sequences are printed on chip as probes,and hybridized with target nucleic acids in tested sample.The results could further be interpreted by computer program or software qualitatively and quantitively.The purpose of this study was to establish a microarray method to screen human pathogenic RNA viruses on genus levels.By using this high throughput method, hopefully,the unknown RNA viral pathogen could be screened to a certain viral genus during the outbreak of viral infectious disease or other public health events.This would be helpful for latter isolation and identification of the pathogen and for further control of the epidemic.The principle of this study was to design the oligo probes on genus levels. For each viral genus,about 10 oligos should be designed to cover all the known viruses in this certain genus.By using these oligos,the known RNA viruses could be quickly identified to its belonged genus.In addition,due to the fact that different viral strains in a certain genus have different sequences but possessed certain amount of same conserved region,by employing long oligos(63mer),certain mismatch of base pairs are permitted.As any new viral strains must have some homology with the sequences in a certain genus,so this method could also be applied for the rapid classification of the novel virus.The chief results of this study are listed as following:1.The designing of oligo probes on genus levels and their validation by bioinformaticsAccording to the database of the eighth report of ICTV submitted in 2005 (http://phene.cpmc.columbia.edu/ICTV/index.htm) and the taxonomy database of NCBI(http://www.ncbi.nlm.gov/Taxomomy/Browser/Viruses),32 RNA viral genus in 14 viral families were selected for this study.Viral genome sequences were downloaded from Genbank.For those genus more than 10 whole genomes were sequenced,all of the whole genome were downloaded.For those less than 10 whole genome were sequenced, apart from downloading the whole genome,other partial sequenced fragments were also downloaded.As for segmented RNA viruses,all of the segments were downloaded. Totally,more than 150000 sequences were obtained for oligo probe designing. Arraydesigner 4.0 and other softwares were employed for the designing.The probe sequenced published by other groups were also taken into consideration.10～30 63-mer oligos were designed for each genus and the Tm value of these oligos were 75±5℃.This was necessary to ensure all the oligos had similar hybridization dynamics.Totally,314 viral probes and 314 complementary probes were designed at the first step.10 positive control probes were designed specifically according to the sequence of Hepatitis C virus. Bioinformatics was employed for the BLAST validation of these oligos.The sequence homology inside the genus and the specificity among different genus were analyzed.2.Selection of oligo probes and the optimization of hybridization conditions Lots of factors could affect the microarray hybridization results.Due to the above reason,after validation of the oligos by bioinformatics,experiments were carried out to validate the specificity of the oligos.The method was to select a virus in a certain genus and to design the specific RT-PCR primers for this virus,and to ensure that the amplified PCR product must contain at least one sequence of the oligo in this genus. Then the PCR product was hybridized with the microarray.By analyzing the hybridization result,the specificity of the oligo probe was evaluated and the other probes hybridized with the PCR product non-specifically was deleted in later experiments.The validation results showed that most of the oligos tested had good specificity,whereas some of the probes continuously gave positive signals.Totally 18 of these probes were deleted by experimental validation.In addition,Japanese B encephalitis virus(JEV) was selected as simulated unknown virus for optimization of hybridization conditions.The nucleic acid was amplified by anchored random-PCR and hybridized with the microarray.Hybridization time,hybridization temperature and concentration of formamide were optimized.The results showed that after printing on chip,the probes were blocked with NaBH4 could decrease the background fluorescence.By comparing the hybridization results under different conditions,hybridization at 42℃for overnight and at 50%formamide in hybridization buffer were selected as the optimal conditions. 3.The exploring of sample pretreatment and target nucleic acids amplification of unknown virusThe interference of host genome and effectively amplification of target nucleic acids from unknown virus were the main obstacles of developing high throughput virus microarray.In this study,the pretreatment of samples by differents ways and different non-sequence relyed amplification methods were explored and compared.The results showed that by sample centrifuge,filtration and treatment with DNaseI/RNaseT1 before RNA extraction,the interference of host genome could significantly be decreased.As for viral nucleic acid amplification,virus-bias primer,random primer and anchored random primer were employed during the reverse transcript of RNA,and random PVA primer PCR,anchored-random primer PCR,mutiple displacement amplification and modified restrict display PCR were compared for their amplification results.The results showed that by using virus-bias primer as reverse transcrip primer and anchored-random primer PCR combined with mutiple displacement amplification for latter amplification,viral nucleic acid could be effectively and balancedly amplified.These pretreatment and optimal amplification could significantly increase the sensitivity and specificity of this microarray.4.The establishment of microarray screening techniqueTaking all the above results together,the medical RNA viruses microarray on genus level was extablished.The testing sensitivity and specificity of the array were evaluated and validated.By testing the cultured supernatants of JEV,the sensitivity of this microarray to JEV was evaluated as about 100 virus copies.Then the cultured supernatants of known viruses and newly isolated sample and simulated clinical sample were used for validation of the microarray's specificity.The results showed that the microarray gave good hybridization results for both cultured cultured supernatants of known viruses and newly isolated sample.For simulated clinical sample,although there existed some non-specific positive singals,the positive singal density of detected virus was higher enough to determine the screening results.In summary,the oligo probes were designed on genus level for human pathogenic RNA viruses and the specificity of the probes was validated both by bioinformatics and experiments.Some of the poor specified probes were deleted.Hybridization conditions were optimized and optimal temperature,time and salt ion concentration were selected for this microarray to ensure the same hybridization dynamics of different oligos.Based on these results,the pretreatment of samples and optimal amplification of unknown virus were also explored.The microarray technique for parallel screening of human pathogenic RNA viruses was primarily established.By using this microarray,the viral pathogens in culture supertatants of both known virus and new isolated sample could be rapidly screened to genus level.Further epxeriments would be taken out to increase the specificity of the microarray and to validate the screen and detect effecicy of the microarray on clinical samples.