Table 3.
SNP analysis at position 109 in NP after passaging four HPAIVs in chicken cells and organs
| Virus | Cells or tissue | Estimated titer of virus with NP 109T mutation at passage no.d: |
||
|---|---|---|---|---|
| 1 | 2 | 3 | ||
| DkYK10 | Chicken braina | >108 | >108 | Not tested |
| Kidney cellsb | ||||
| Embryoc lung | >107 | >106 | >107 | |
| Embryo brain | >108 | >108 | >107 | |
| CkYM7 | Embryo lung | >106 | ||
| Embryo brain | >105 | >106 | >107 | |
| CkMZ11 | Embryo lung | |||
| Embryo brain | >107 | >107 | >107 | |
| WsAK1 | Embryo lung | |||
| Embryo brain | >106 | >106 | ||
Ten percent lung homogenate (L1) was prepared from a dead chicken after inoculation with DkYK10 and intranasally inoculated into chickens. The brain (B1) was harvested from the first dying chicken, the supernatant of the 10% brain homogenate was inoculated intranasally into another 5 chickens, and B2 tissue was obtained.
The allantoic fluid infected with DkYK10 was inoculated onto primary chicken kidney cells at an MOI of 0.01 and incubated for 2 days at 37°C. The culture fluid was passaged another two times in the cells.
Seventeen-day-old embryonated chicken eggs were inoculated with HPAIV (106 EID50) in the allantoic cavity, and lung or brain tissues were harvested after 48 h of cultivation. The 10% lung or brain homogenates were then passaged into the allantoic cavities of the embryonated eggs to obtain the respective lung or brain passage viruses.
Virus RNA was purified from tissue samples and tested for the emergence of the NP-109T mutation using real-time PCR based on SNP analysis. The average SNP value (45 − CT) of 3 wells was determined. The 106 EID50 of DkYK10 was mixed with 103, 104, 105, or 106 EID50 of DkYK10-B5, and the SNP values of these mixtures were used as a standard to estimate titers of virus (EID50/g tissue) with the NP 109T mutation.