Molecular Epidemiological Investigation Of Newcastle Disease Virus Isolates From Guizhou Province And Real-time RT-PCR Detection

Newcastle disease(ND) is regarded worldwide as one of the most devastating diseases of poultry.Its causative agent.Newcastle disease virus(NDV),is classified as a member of the order Mononegavirales family,Paramyxovirinae subfamily, Paramyxovirinae genus rubulavirus.Because of the severe nature of the disease and the associated consequences,ND is included as an Office International des Epizooties(OIE) list A disease.In the past two decades,an intensive vaccination program against ND has been practiced in both large-scale poultry operations and village poultry farming.Disease outbreaks occur infrequently in some vaccinated flocks,however,infections of atypical Newcastle disease in the chicken flocks have been more and more frequently reported since the late 1990s.Even some chicken flocks with high antibody level can also suffer ND.Some scholar think that the emergence of new genotypes or subgenotypes could be responsible for ND outbreak in vaccinated flocks.Especially when the emergence of variant NDV of goose origin was found,people are more and more interest in mechanism of the atypical Newcastled isease caused by virulents train.The fragment containing Fusion(F) gene of the 8 representative Newcastle disease virus(NDV) isolated from Guizhou current outbreak ND poultry farms between 1999-2004 were amplified by reverse transcriptionpolymerase chain reaction(RT-PCR) technique.F gene were sequenced after the products of amplification being cloned. Compared with the published sequences of representative NDV strains,On of these studies,we can see more information about the nucleotide.It can give great help for the study of the prevalence condition of NDV.There are several kinds of classical NDV detection methods,such as virus isolation, HA and HI test,immunofluencent test,ELISA,etc.These techniques some are time-consuming,some are insensitive or somehow unspecific.Common RT-PCR can detect a few copies of nucleic acids of NDV,and therefore it is used to detect NDV for clinical or quarantine purpose.But its uses are quitely limited because it is dificult to avoid the false positive signals caused by the pollution of PCR products.Common RT-PCR also requires the uses of EB,a strongc arcinogen. Real-time RT-PCR assay is one of the new detectionte chniques.It has high sensitivity as common RT-PCR,and avoids both the pollution of PCR products and the uses of EB.It is also faster than common RT-PCR.SYBR Greenâ… releases fluorescence when combining to double-stranded DNA,and based on this principle,SYBR Greenâ… real-time RT-PCR is applied widely with lower cost and without dificult probe design.It is suitable for the detection of highly variable genes.Partâ… :Cloning and Sequence Analysis of F Gene of Newcastle disease virus Isolated from Guizhou Province One pair of specific primers were designed and synthesized according to fusion(F) protein gene sequences of Newcastle disease virus (NDV)from GenBank,The fragments of F gene of 8 NDV strains isolated from Guizhou province were amplified by RT—PCR,then the amplified fragments were cloned into pMD—18T vector and the recombinant plasmids were sequenced.The results showed that the F gene from all of the NDV isolates consisted of 1 662 bp,coding for 553 amino acid,Neucleitide and amino acid sequence homologies were found to be 84.0%—99.7%and 87.5%—99.3%respectively;but the amino acid sequence homologies to other NDV strains such as LaSota,B1 and F48E9 were only from 87.2%—97.7%.The deduced amino acid sequence of the cleavage site of F protein of the NDV isolates were ¹¹²R—R—Q—K/R—R—F¹¹⁷(7 strains were virulent) and ¹¹²G—R—Q—G—R—L¹¹⁷(1 strains was avirulent).The NDV systemic evolution tree was depicted by MegAlign soft-ware and showed that the four strains were gene typeâ…¦,the two strains were gene typeâ…¡,And two strains were gene typeâ…¨.Partâ…¡:Pathotyping of Newcastle Disease Virus by SYBR Greenâ… Rea1-Time RT-PCR the virulence of Newcastale disease virus(NDV) strains was determined by the lysis site sequences of the viral F protein.Accordingly,A pair of primers were designed for pathotyping of Newcastale disease virus(NDV).8 sWains of NDV were amplified using the primers respectively by one-step SYBR Greenâ… RT-PCR,and the virus was pathotypied with the amplification effects of the a pair of primers and the Tm values of the amplification products.Pathotyping results of 8 strains of this assay were as the same as those of Mean Death Time(MDT)and sequencing.At the same time,80 samples were amplified by three different methods,the results of SYBR Green I RT-PCR were compared with those of conventional RT-PCR and embryonated eggs.The results showed that the assay was sensitive,specific and rapid enough to pathotype NDV strains.Partâ…¢:Detection of distribution of virulent Newcastle disease virus in chicken by real-time RT-PCR The real-time RT-PCR methold was applied to detect Newcastle disease virus(NDV) virulent H₂ strain in 18 day-old chicken infected artificially with NDV and infected naturally with NDV,And the results indicate:(1) lung,brain,spleen,kidney,duodenum were positive 6h post infected(PI) by nose and eye dripping and all samples were positive 12h PV。(2) 6h PI naturally,All tissue were negative,but lung,brain,spleen,kidney,duodenum,liver werepositive 12h PI.All samples from chicken were positive 24h PI(3) Two experimental results indicate that all samples with virus content from high to low in order are:lung,brain,spleen,kidney, duodenum,liver,heart,leg muscle.At the same time,doing a sevsitive comparison of the real-time RT-PCR assay and common RT-PCR assay...