[Generation of newcastle disease virus strain ZJI isolated from an outbreak in the goose using reverse genetics technique].
The full-length cDNA clone, NDV3GM122, and the three helperplasmids pCI-NP, pCI-P and pCI-L of Newcastle disease virus strain ZJI isolated from an outbreak in the goose were cotransfected into BSR-T7/5 cell expressing T7 RNA polymerase. Meanwhile, the full-length cDNA clone NDV3GM122 and the three helperplasmids, pCIneoNP, pCIneoP and pCIneoL which were derived from NDV strain La Sota, were also cotransfected into the cell, respectively. Indiect immunofluorescence assay (IFA) was performed 48 to 96 hours post-transfection using NDV HN-specific monoclonal anbtibody (McAb) 6B1 and bright stainings were found in the transfectants, indicating that the full-length clone was functional and the HN protein was expressed. The transfected cell and the supernatant were mixed well and thereafter the mixture was inoculated into specific pathogen free (SPF) chicken eggs. The allanotoic fluid of the injected eggs gave a positive hemagglutinin( HA) titer ranging from 16 to 32 in the secondary passage and increased to 128 in the third passage, which was same to the level of parent wild-type virus. The allantoic fluid containing the recovered NDV was analyzed in hemagglutination inhibition( HI) test by using McAb 6B1 and the specific inhibition was found. The typical morphology of the produced NDV was detected in the electronic microscope. The results mentioned above demonstrated that infectious NDV of strain ZJI was successfully generated, which laid good foundation for the further related research.
Liu
Yu-liang
YL
Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, China.
Zhang
Yan-mei
YM
Hu
Shun-lin
SL
Wu
Yan-tao
YT
Liu
Xiu-fan
XF
Long
Jin-xue
JX
Shi
Huo-ying
HY
Zhang
Xiao-rong
XR
Zhang
Ru-kuan
RK
D000818:Animals; D002642:Chick Embryo; D004196:Disease Outbreaks; D019084:Fluorescent Antibody Technique, Indirect; D005777:Geese / Q000821:virology*; D005821:Genetic Techniques; D009521:Newcastle Disease / Q000453:epidemiology*; D009522:Newcastle disease virus / Q000235:genetics* / Q000302:isolation & purification; D011201:Poultry Diseases / Q000453:epidemiology* / Q000382:microbiology; D014162:Transfection