Development And Implementation Of Multiplex RT-PCR For The Rapid And Differential Diagnosis Of Duck Hepatitis Virus

Duck virus hepatitis (DVH) caused by duck hepatitis virus (DHV), is an acute, rapidlyspreading, and fatal disease occurred among young ducklings within three weeks, thepathological change main in liver. According to the Virus Taxonomy tenth Report of theInternational Committee on Taxonomy of Viruses (ICTV), DHV was classified into threeserotypes: duck hepatitis A virus (DHAV), duck astrovirus type1(DAstV-1) and duckastrovirus type2(DAstV-2). At the same time, DHAV could be classified into three genotypesor subtypes: DHAV-1, DHAV-2and DHAV-3. No obvious antigenic relationships have beenfound among the three serotypes of DHV and the three genotypes of DHAV by the serumneutralization test, it is difficult to control and cure DVH in ducklings. DHAV-1was alwaysthe superiority serotype in China, but DHAV-3was also widespread in recent years,co-infection of DHAV-1and DHAV-3was found in some areas even, the occurrence ofDAstV-1in China was further increased the difficulty of DHV prevention and control. In thisstudy, we established a multiplex RT-PCR assay for the rapid and differential diagnosis ofDHV, and detected clinical samples from Shangdong, Guangdong, Sichuan, Henan fourprovinces in2011, and clinical samples from Shandong province in2012. This study consistsof three parts as follows:1. The isolation and complete gene sequencing and analysis of one DAstV-1strain WF1201According to the whole genome sequence alignments of DAstV-1sequences retrieved fromGenBank, we selected two pairs of detection primers in the conservative region of DAstV-1and established corresponding RT-PCR assays. Apply this two pairs of detection primers todetect twenty-four samples from Sichuan and Shangdong province. We detected nineDAstV-1-positive samples. DAstV-1-positive samples were pestled and infected to ninth-dayold duck embryos and one-day old ducklings. No duck embryos died and no obviouspathological changes in embryos except the allantoic fluid changed into green or kelly. Wesuccessfully detected DAstV-1from all infected duck embryos by the RT-PCR assay. Therewere20%infected ducklings appeared neurological sign within48hours, then died. We can see visible hepatomegaly and haemorrhage. Others were feed one week and none died.According to the published complete genome sequences of DAstV-1in GenBank, we selected10pairs of overlapping primers and two pairs of Reace-PCR primers to amplified thecomplete gene of strain WF1201. Sequencing result showed that the gene had all typicalcharacteristics of DAstV：there is a heptameric AAAAAAC sequence of ribosomal frameshiftand a stem–loop sequence in the overlap region between ORF1a and ORF1b. Sequencingresult indicated that the strain WF1201had close genetic relationship and a high nucleotidehomology of98.7%with the strain C-NGB isolated from Jiangsu province. Transmit thesequence to GenBank, and the accession number is JX439643.2. Improved duplex RT-PCR assay for differential diagnosis of mixed infection of duckhepatitis A virus type1and type3in ducklings.According to the whole genome sequence alignments of72DHAV-1strain sequences and20DHAV-3strain sequences retrieved from GenBank, we selected two pairs of primers forDHAV-1and DHAV-3in the conservative region. Applied this two pairs of primer, weestablished two simplex RT-PCR assays to detect DHAV-1and DHAV-3respectively, thenwe detected the specificity of the primers. After optimized the reaction condition, a duplexRT-PCR assay was established based on the two simplex RT-PCR assays. This assay hadstrong specificity, only had amplification specific to DHAV-1and DHAV-3, no amplificationoccurred using nucleic acids from other duck pathogens as AIV(H9N2), NDV, DPV, DuCV,RA and E. coli. The minimum detection assay showed the method had a high sensitivity, theminimum detection limit of the method has been determined to be10pg total RNA templatesextracted from duck liver samples or102copies viral RNA of DHAV-1and DHAV-3respectively. Using the method,60clinical liver samples from26duckling flocks inShandong, Guangdong, Sichuan and Henan province of China were detected. Among them,23（38.3%）were mixed infection of DHAV-1and DHAV-3,11（18.3%）samples were DHAV-1single infection,18（30%）samples were DHAV-3single infection, and8（13.3%）sampleswere negative of DHAV-1and DHAV-3.3. Development of a multiplex RT-PCR assay for detection of duck hepatitis A virus type1，type3and duck astrovirus type1According to the whole genome sequence alignments of DAV-1, DHV-3and DAstV-1 sequences retrieved from GenBank, we selected three pairs of primers for DHAV-1, DHAV-3and DAstV-1in the conservative region. Applied this three pairs of primer, we established threesimplex RT-PCR assays to detect DHAV-1, DHAV-3and DAstV-1respectively, then wedetected the specificity of primers. After optimized the reaction condition, a multiplex RT-PCRassay which could detect the three strains at the same time was established. This assay only hadamplification specific to DHAV-1, DHAV-3and DAstV-1, no amplification occurred usingnucleic acids from other duck pathogens such as AIV(H9N2), NDV, DPV, DuCV, RA and E.coli. It had strong specificity. The minimum detection limit of the method has been determinedto be10pg total RNA templates extracted from duck liver samples or102copies viral RNA ofDHAV-1and DHAV-3respectively. The method, for the first time, made the rapid differentialdiagnosis of all DHV subtypes occurred in China in one step come true. Using the method,90clinical liver samples from Shandong, province of China were detected. There were48（53.3%）samples died by DHV infection. Among the48samples,8samples were mixed infected byDHAV-1and DHAV-3,3samples were mixed infected by DHAV-3and DAstV-1.11sampleswere infected by DHAV-1,18samples were infected by DHAV-3and8samples were infectedby DAstV-1.