Investigation Of In Vivo Distribution Of Duck Hepatitis A Virus Type 1 And Type 3 In Clinical Dead Ducklings

1. Development of duplex real-time TaqMan PCR assay for DHAV-1 and DHAV-3Duck hepatitis virus(DHV), an acute highly contagious disease of ducklings caused by duck hepatitis A virus(DHAV) is distributed worldwide. The infected ducklings assumed obvious opisthotonos characterized with enlargement and hemorrhage of liver. DHAV can be divided into three genotypes: DHAV-1, DHAV-2 and DHAV-3, and DVH disease in our mainland is mainly caused by DHAV-1 and DHAV-3. Due to the similar clinical symptoms and pathological changes of the infected ducklings, it’s very hard to distinguish the infection of the two viruses merely by clinical diagnosis. Besides, there are co-infection in duck industry which makes the prevention and treatment harder than before. The study has established a duplex real-time PCR assay for DHAV-1 and DHAV-3 detection, and has investigated the epidemiology in Shandong province and the clinical in vivo distribution of DHAVs ducklings. The study includes two parts:According to the results of sequence alignment of DHAV-1 and DHAV-3 by MEGA 4.0,two pairs of primers and two TaqMan TM probes were designed in the conserved region. The probes were tabbed with CY5/BHQ2 and FAM/TAMRA, respectively. After the optimization of conditions, a duplex real-time PCR was established. Using 10-fold serially diluted standard linear templates, the standard curves for DHAV-1 were generated with a range from 6.7×107to 6.7×103 gene copies per reaction. The standard curve for detecting DHAV-1 is YDHAV-1 =-3.2850x+40.812, with linear correlation coefficient 0.998 and reaction efficiency 101.5%.The standard curves for DHAV-3 were generated with a range from 1.1×107 to 1.1×102 gene copies per reaction, and the equation is YDHAV-3 =-3.2791x+39.216, with linear correlation coefficient 0.999 and reaction efficiency 101.8%. The minimum detection limits of the duplex real-time PCR assay were 14 copies of DHAV-1 cDNA and 22 copies of DHAV-3 cDNA per reaction, which were equivalent to the simplex real-time PCRs. The intra-assay coefficients of variation(CVs) for detection of the DHAV-1 and DHAV-3 standard templates ranged from0.28% to 1.24% and from 0.28% to 1.81%, while the inter-assay coefficients of variation were from 0.29% to 0.79% and from 0.22% to 0.72%, respectively. The duplex real-time PCR assay was able to detect the viral cDNAs of DHAV-1 LY0801 strain, DHAV-3 SD1201 strainand ten positive liver samples for DHAV-1 and DHAV-3, with no cross-reaction between DHAV-1 and DHAV-3. Under the same condition, no amplification occurred using nucleic acids from the 7 other duck pathogens.Thirty artificially infected duckling livers and 200 suspected DVH duckling livers were tested by the duplex real-time PCR and conventional RT-PCR. The results showed that the positive rates of artificially infected samples for DHAV-1 and DHAV-3 by duplex real-time PCR and conventional RT-PCR were all 100%. For the clinical samples, the positive rates of DHAVs was DHAV-1 and DHAV-3 were 29.0% and 56.5% by the duplex real-time PCR assay.Moreover, among the 200 clinical samples, there were 24(12%)samples confirmed co-infection positive by the duplex real-time PCR. The positive rates tested by real-time PCR were higher than conventional RT-PCR, the results indicated that the duplex real-time PCR assay was specific and sensitive, and it can be used in quick diagnosis and distinguishing of DHAV-1 and DHAV-3 as well as the quantitative detection of DHAVs in infected ducklings.2. The distribution of DHAV-1 and DHAV-3 in different organs of clinical dead ducklingsThe organs of clinical DHAV-positive dead ducklings were collected and tested by duplex real-time PCR assay, including heart, liver, spleen, kidney, thymus, Bursa of Fabricius,brain and pancreas. The results showed that DHAV can be detected all the inspected tissues with different viral load in different tissues, which indicated that the tissue tropism of DHAV in ducklings is broad. The most obvious pathological changes of livers, spleens and kidneys in dead ducklings in clinical diagnosis, assuming enlargement, hemorrhage and necrosis.According to the results of duplex real-time PCR assay, the viral load of DHAVs was the highest in liver and spleen. The viral load of DHAV-1 in liver, spleen and kidney were up to1012.32 and 1011.23copies/g respectively, and the viral load of DHAV-3 were up to 1012.07 and1011.63copies/g respectively. The thymus and pancreas assumed hemorrhage, and the Bursa of Fabricius was swollen and some brains assumed edema and liquefactive necrosis. DHAV can be detected in all the organs with a lower viral load. By independent samples T test, the viral load in the same organs showed no difference, suggesting that coinfection of the two viruses has no effect on the late stage of virus replication. Most clinical dead ducklings aged from5~14 days old, and there is no correlation between in vivo viral load and the age of theducklings. The results above showed that the extent of pathological change of tissues has positive correlation with the viral load, and liver, spleen and kidney is the ideal target organs for proliferation of DHAVs. The results has laid a theoretical foundation for the study of pathogenesis of DHAV.