Development Of DNA Microarrays For The Detection Of Mosquito-borne Viruses And Influenza Viruses

BackgroundMosquito-borne viruses are spreaded by mosquitos to the thermostat vertebratesincluding: Dengue virus, Japanese encephalitis virus, yellow fever virus, West Nilevirus, and Chikungunya virus, etc. Some of the mosquito-borne viruses have beenclassified as biological warfare agents. In addition to the viruses, malarial parasite andlymphatic filariasis, carried and transmitted by mosquito, are the most prevalentparasitic diseases in the world. Malaria and lymphatic filariasis are listed as thehighest global priority diseases to be controlled and eliminated due to their wideprevalence and serious hazards. The emergency spread ability of Mosquito-bornepathogens in new habitats make great significance to continuous monitoring thesepathogens. In China, the mosquito-brone pathogens are significant pathogens intropic, grass, and forest war zones, and seriously affect the battle ability of militarydue to these diseases. Disease prevention and control are constituted by diagnosis,prevention, and treatment. Among them, diagnosis is not only indispensable fordisease control, but also a prerequisite for effective prevention.Influenza viruses are divided into three types: A, B, and C. Presently, there aretwo categories of first-line influenza antiviral drugs used in clinical settings:neuraminidase inhibitors and matrix protein2-ion channel blockers. Drug resistanceof Influenza virus on these drug were obeserved in the past years. Resistancedetection of influenza virus is significance for appropriate treatment and prevention,and regulating the use of antiviral therapy. In2013, the suddenly appeared Avianinfluenza A (H7N9) induced great panic in China. By the second week of2014, therewere210laboratory confirmed infection patients in China, and the mortality rate is upto22%. Many studies have shown that it is effective to administrate neuraminidaseinhibitors within48h of influenza symptom onset. Therefore, rapid and accuratediagnosis on avian influenza A (H7N9) and other common influenza virus areimportant for clinical treatment and prevention.Therefore, in this study, three DNA microarray kits were developed for thedetection of primary mosquito-borne viruses and parasites, oseltamivir andamantadine resistance of common influenza A viruses, avian influenza A (H7N9) andother common influenza viruses. These microarray kits provided new techniques toimprove the urgent detection ability of these biological warfare agents and epidemic for the military army in tropic and forest war zones.MethodsTechnical route includes three steps.1, Screen primers and probes. Targetpathogens and target genes are determined by literature search. Primers and probes aredesigned for microarray. Pathogens were collected for establish standard plasmid,while artificial synthetic target genes are prepared for other deficient target pathogens.Sensitivity and specificity references were prepared and primers and probes werepreliminary screened.2, Experimental conditions optimization. The multiplexRT-PCR, multiplex PCR systems, and microarray detection methods were optimized.3, Evaluate microarray kits. The specificity of microarray kits are evaluated bypositive references and negative references. The sensitivity of microarray kits areevaluated by sensitivity references or compared with other reference detectionmethods.4, actual specimens are detected by the microarray kits.Results1.Development of DNA microarray for primary mosquito-borne viruses andparasites detection. First, Target pathogens were determined by literature search,which include twelve of primary mosquito-borne viruses (dengue virus1-4, Japaneseencephalitis virus, yellow fever virus, St. Louis encephalitis virus, West Nile virus,Eastern equine encephalitis virus, Western equine encephalitis virus, and Venezuelanequine encephalitis virus) and seven of common mosquito-borne parasites(Plasmodium vivax, Plasmodium malariae, Plasmodium falciparum, Plasmodiumovale, Wuchereria bancrofti, Brugia malayi, and Brugia timori). Plasmid of twelve ofviruses and7of parasites were established. In vitro transcribed RNA of10viruseswere transcribed by the plasmid templates as sensitivity reference. Virus-like particles,constructed by MS2bacteriophage, were established as the RNA positive referencesof the mosquito-borne virus microarray. Second, nineteen primers and21probeswere screened for the amplification and detection of mosquito-borne viruses andparasites. Third, Amplification systems were optimized to ensure that all of the targetgenes were amplified. The mosquito-borne viruses were divided into two group ofmultiplex RT-PCR, as well as the parasites were establish a multiplex PCR.Chemiluminescence detection method was established and optimized for themicroarray. Finally, the performance of the microarray was evaluated. Specificityevaluation results show that the microarray can distingusih these twelve viruses and six parasites (B. malayi and B. timori could not be subtyping). Sensitivity evaluationresults show that the sensitivity limitations of the mosquito-borne virus microarraywere102-103copies/reaction, and the detection limitations of mosquito-borne parasitemicroarray kit were101-103copies/reaction. The detection limitation of JEV was100.8-101.8PFU/ml as performed on one JEV sample, which was derive from ChinaCDC.2. Development of visualization DNA microarray for primary influenza A virusresistance detection. A cost-effective, rapid, and high-throughput oligonucleotidemicroarray visualization method, which based on quantum dot-catalyzed silverdeposition, was developed for the simultaneous detection of neuraminidase H275Yand E119V; matrix protein2V27A and S31N mutations of influenza A (H3N2),seasonal influenza A (H1N1), and2009influenza A (H1N1). First, plasmids ofoseltamivir and amantadine-susceptible and resistant genotypes were established andin vitro transcribed RNA were also transcribed. Second, two pairs of primers andtwenty-three probes were screened for the detection of amantadine resistancemutations and combined with oseltamivir primers and probes which derived fromliterature. Third, two group of multiplex RT-PCR and the visualization detectionmethod, which based on quantum dot-catalyzed silver deposition, was established andoptimized. Fourth, the performance of the microarray was evaluated. The sub-typingspecificity of the microarray was evaluated by positive strains of influenza and apanel of negative controls. In vitro transcribed RNAs of oseltamivir andamantadine-susceptible and resistant genotypes were also used as templates todetermine the reliability of genotypes detection. The results showed the microarraypossessed similar detection sensitivities as the influenza real-time RT-PCR kit. Thesensitivity of the method to distinguish the mixed population of the drug-sensitive andresistant was compared by detection of the mixed in vitro transcribed RNAs templatesof PH1N1, and the results showed that the detection limitation of the minorpopulation was1%-5%. Finally, the detection results of307clinical throat swabsamples showed that all the281positive samples resistant to amantadine (S31N), andall of the five seasonal influenza A (H1N1) samples were resistant to both oseltamivir(H275Y) and amantadine (S31N), none of the influenza A (H3N2) and2009influenzaA (H1N1) samples were resistant to oseltamivir. The consistence between sequencingand microarray was98.8%.3. Development of DNA microarray for the new emergence avian influenza A (H7N9) detection. A microarray detection method was developed to subtype avianinfluenza A (H7N9), avian influenza A (H5N1),2009influenza A (H1N1), seasonalinfluenza A (H1N1), and seasonal influenza A (H3N2). The microarray can universaldetected influenza A virus and B virus at the same time. First, two pairs of primersand three probes of HA and NA genes of H7N9were screened as well as one groupprimer and probe of human RNase P. Second, these primes and probes were combinedwith that of other influenza viruses which derived from literature. Three set ofmultiplex RT-PCR systems and chemiluminescence detection method wereestablished and optimized. Third, forty culture influenza viruses were detected todetermine the subtyping reliability for these influenza viruses. The results ofsensitivity evaluation show that the sensitivity of the microarray was similar orten-fold lower than influenza real time RT-PCR kits. Finally, the detection results ofsixty-six of clinical throat swab samples verified the effectiveness of the microarray.ConclusionsThree sets of microarray kits were development to detect primarymosquito-borne viruses and parasites, oseltamivir and amantadine resistance ofprimary influenza A viruses, subtypes of the new emergence of avian influenza A(H7N9) and other primary influenza viruses. Specificity and sensitivity evaluationresults showed that performance of the three microarray kits achieved the designedparameters. This study provided new methods for the detection of primarymosquito-borne viruses and parasites, resistance and genotyping of common influenzaviruses. These microarray kits provided new techniques to improve the urgentdetection ability of these biological warfare agents and epidemic for the military armyin tropic and forest war zones.