Isolation And Identification Of Duck

The liver tissue samples of infected ducks from Southwest China were detected in this study. Afterwards, the suspected virus was isolated and identified, then, the sensitivity of this virus to duck embryo fibroblast mono layer cells and ducklings were evaluated. What is more, the complete genome of this virus was sequenced and analyzed. The main content and results are shown as follows.1. Isolation and Identification of the provisional virus. The tissue samples were undergone excision, homogenization, filtering, and then inoculated into allantoic cavites of 9 day-old duck embryonated eggs. After three times passage, the duck embryonated eggs infected by this virus were died within 4 days and were observed severely swollen and hemorrhage. Similar to other flavivirus, it was a RNA virus, what is more, the identification results suggested that it was an enveloped virus and were sensitive to ether, chloroform. Other pathogens that cause similar symptoms such as DPV, DuCV, DHBV, AIV, were negative for PCR or RT-PCR. Primers were designed based on the NS3-NS4 genome partial of TMUV-WR isolate to detect TMUV. The prospective stripe was acquired, after sequencing, the identity of this partial was found to be 72.3% with Ntaya virus (IPDIA isolate). The highest and lowest identity of Chinese isolates were found to be 99.4%,96.8%, comparing with GX2013H isolate (GenBank NO:KJ700462) and 1q-1 isolate (GenBank NO:KF557893), respectively. Based on the results of our study, a novel TMUV isolate, separated from a duck in southwest China, afterwards was named CQW1.2. Pathogenic features of CQW1 in DEF cells and ducklings. After five passages in embryonated duck eggs, the harvested allantoic fulid was inoculated into duck embryo fibroblast (DEF) cells. Cytopathic effects (CPE) were observed daily and typical (CPE) was observed in DEF cells at 24-72 h after inoculation characterized by vacuoles degeneration, necrocytosis, rupturing, indicating that CQW1 strain can adapt to DEF cells and cause CPE immediately. The TCID50 and ELD50 were found to be 1055 TCID50/0.05 mL and 102.75 ELD5o/0.2 mL, respectively. Animal experiment was performed in twenty 7-day-old healthy Cherry Valley ducklings. Ten ducklings were infected by intravenous injection with CQW1 virus stock (Containing 102.75 ELD50) and the rest ten ducklings were received PBS for control. Results showed that effected ducklings showing clinical sign characterized by loss of appetite, lassitude, ataxia, paralysis. Some ducklings were unable to stand steadily and falling. The morbidity and mortality of ducklings were as high as 100% and 60%. The results of RT-PCR detection showed that positive for TMUV in blood, liver, brain of infected ducklings. Histopathological studies showed that infected ducks had systemic lesions. The morphological alteration of neuronal nuclei, neuronophagia and microglial nodule were observed in the brain tissue of inoculated ducklings, additionally, the liver tissue were affected mainly proved by disordered lobular architecture, degeneration, necrosis and regenerated hepatocytes.3. Sequencing and phylogenetic analysis of CQWl. Based on the primers overlapping which covering the whole genome of TMUV, the full genome of CQW1 was amplified (GenBank NO. KM233707). Genome analysis suggested that full-length genome of CQW1 was 10992 nt with a single ORF extending from 96-10373 nt, encoding a 3425 amino acid (aa) polyprotein. It was found that CQW1 strain shared the same genome organization with other TMUV strains. The evolutional trees constructed indicated that CQW1 strain and other TMUV clustered together and formed novel taxa of Ntaya group. Additional, CQWl strain was most closely related to GX2013H and they formed a separate clade that was distinct from other TMUV strains. What is more, the average distance between CQW1 strain and the other 40 members was 0.0299 (mean distance 0.0163 within 40 members), indicating that CQW1 strain unequivocally contributed to the regional divergence of TMUV. Four groups based on different year including 34 TMUV E genes were selected and analyzed. Analyses of viral genetic diversity showed a relatively high increase over time and the E gene of TMUV has evolved at approximately 3.8 × 10 substitutions/site/year. Additionally, the minimum nucleotide diversity among different years was found to be 2010 and the highest was 2013, it was showed that the nucleotide diversity during 2011-2012 were relatively stable. Our results of diversity analysis showed the genetic diversity characteristic during 2010-2013.