Rapid Detection Method Of Type 3 Duck Hepatitis A Virus And Study On The Infection Of Young Ducks

To explore adaption of Duck hepatitis A virus 3 （DHAV-3） to growth in cell cultures and its proliferation, quantitative detection method for DHAV-3, and the proliferation, biodistribution and excretion rule of DHAV-3 in young duck after artificial infection in this paper, obtained the following results:1. Adaption of DHAV-3 to growth in cell cultures and its proliferationAfter DHAV-3 four blind passages in DEF and three blind passages in DELC, respectively, the obvious and continuous cell lesions were observed. DHAV-3 could be detected after 4 h post infection in DEF by using qPCR. The copy number of DHAV-3 in DEF showed a rising trend with the passage of time, and reached the maximum at 72 h (10^5.76copies/μL). DHAV-3 could be detected after 4 h post infection in DEF by TCID₅₀. The titer of DHAV-3 in DEF indicated that the trend of continuous increase with the elapse of time, and reached the maximum at 72 h (10^4.42/100μL). DHAV-3 could be detected after 4 h post infection in DELC by qPCR. The copy number of DHAV-3 in DELC showed a rising trend with the extension of time, and reached the highest at 60 h (10^7.14 copies/μL). DHAV-3 could be detected after 4 h post infection in DELC by TCID₅₀. The titer of DHAV-3 in DELC indicated that a trend of continuous increase with the passage of time, and reached the maximum at 60 h (10^5.75/100μL). DHAV-3 in the supernatant of cell cultures were lower than in DEF and DELC.2. Improved SYBR Green I quantitative PCR （qPCR） assay for detection of DHAV-3Based on the VP3 gene of DHAV-3, a pair of specific primers were designed for development SYBR Green I quantitative PCR （qPCR） detection method. The standard equation was Y=-3.317 X+36.434, and the correlation coefficient was 0.996, while the template concentration was between 10² copies/μL to 10⁹ copies/μL. The melting curve with a single specific peak for DHAV-3. The detection results were negative when other related duck pathogens （Duck hepatitis A virus 1, Duck plague virus, Escherichia coli, Salmonella enteritidis, Riemerella anatipestifer） were detected by qPCR. The minimum detection limit of the assay was 10² copies/μL. It showed excellent specificity and sensibility. Established qPCR and TCID₅₀ were used to detect the proliferation rule of DHAV-3 in DELC and DEF, respectively. And the results showed the proliferation tendency were coincident.3. Developing real-time fluorescent reverse transcription loop-mediated isothermal technology （real-time RT-LAMP） detection method for detection of DHAV-3Three pairs of specific primers were designed based on the VP3 gene of DHAV-3, and a simple and rapid real-time fluorescent reverse transcription loop-mediated isothermal technology （real-time RT-LAMP） which was used to detect DAHV-3 was established. There was no cross-reactivity between DHAV-3 and other related pathogens （Duck hepatitis A virus 1, Duck plague virus, Riemerella anatipestifer, Salmonella enteritidis, Escherichia coli）. The minimum detection limit of the real-time RT-LAMP method was 2.4×104 copies/μL. It showed excellent specificity and sensibility. Real-time RT-LAMP was an effective method for detection of DHAV-3 since it could meet the requirement of qualitative and quantitative detection.4. The proliferation, biodistribution and excretion rule of DHAV-3 in young duck after artificial infectionAmong 60 negative young ducks of 2 months,55 ducks were infected artificially by intramuscular injected with the allantoic fluid of DHAV-3 at dose of 10¹⁵ copies of each duck （QL strains）,5 ducks were selected and dissected in each time point after infection at Id,2d,4d,7d,14d,21d,28d,42d,56d and 70d, then collected respiratory organs, immune organs, digestive organs and intestinal contents, etc. The copy number of DHAV-3 was detected by using qRT-PCR in the collected organization and contents.5 ducks as blank control group, the blood was collected for detection of DHAV-3 at each time point. The results were as follows:（1） The proliferation rule:the copy number of DHAV-3 in liver showed a rising trend after 1d-56d post infection, and reached the highest at 56d post infection with the copy number of DHAV-3 was 10⁹⁴¹ copies in the 0.1 g liver, which was the highest level of virus in all collected organs.（2） The distribution rule:DHAV-3 in all the collected organization were detected except for the trachea. DHAV-3 content in the most organs showed a trend of continuous proliferation with the extension of time. DHAV-3 content in the lung, spleen, duodenum, brain, heart and blood reached the maximum number after 56d post infection, which the copy number of 10^7.86 copies,10^9.19 copies,10^8.42 copies,10^8.71 copies,10^8.85 copies,10^7.09 copies, respectively, the virus copy number declining after at 70d post infection. The proliferation of DHAV-3 reached a peak after infection at 70d in the bursa of fabricius, esophagus, muscular stomach, glandular stomach, jejunum, ileum, cecum, rectum, kidney and muscle, which the copy number was between 10^7.57 copies and 10^8.49 copies. In these collected organs, DHAV-3 has the highest levels in the spleen when reached the peaks, which was higher than other organs.（3） The excretion rule:the trend of DHAV-3 content increased gradually after 1d-70d post infection in the oral cavity, gastric contents, intestinal contents and cloaca contents, reached the highest at 70d post infection with the copy number of 10^5.61 copies-10^6.92 copies. The copy number of DHAV-3 in the gastric contents was minimum and in the jejunum contents was maximum.5. Comparing three methods of detecting for DHAV-3Thirty clinical liver samples were detected at the same time by using establishment of the real-time RT-LAMP and qPCR in the study, and RT-PCR assays. The results that the sensitivity, specificity and coincidence rate were similar for real-time RT-LAMP and RT-PCR. The real-time RT-LAMP method showed 84.21% sensitivity,100% specificity and a 90% coincidence rate when compared with qPCR. Firstly, qRT-PCR and qPCR only needed a pair of primers, and real-time RT-LAMP needed three pairs of primers. Secondly, qRT-PCR and qPCR took 90 to 120 minutes to obtain the results and real-time RT-LAMP only took 50 minutes. Besides, the specificity and sensitivity of qRT-PCR was best of these three assays.