Virulence Evolution Of ClassⅠ Newcastle Disease Virus And The Role Of Autophagy In NDV Infected Tumor Cells

Newcastle disease (ND) is one of the most serious avian diseases. ND can cause substantialeconomic losses and remains a major threat to the poultry industry worldwide. The causative agent ofthe disease is Newcastle disease virus (NDV), also designated as avian paramyxovirus serotype1(APMV-1), an enveloped virus belonging to the genus Avulavirus within family Paramyxoviridae.Phylogenetic analyses have revealed that NDV strains consist of two distinct classes (I and Ⅱ) within asingle serotype, with class I containing nine genotypes (1–9) and class Ⅱ containing eleven genotypes(I–XI). Since1926, ND has caused four pandemics worldwide and class Ⅱ isolates being responsible forall four outbreaks. Given that the existence of class I NDV is more widespread than previously thoughtassessing the biological characteristics of class I NDV, as well as determining their antigenicdifferences with class Ⅱ NDVs, may be helpful in our understanding for the control and prevention ofND in poultry.In the present study we have sequenced the complete genome of a new lentogenic class I isolate,JS10, and its genetic and antigenic properties compared with those of La Sota. On the basis of theNDV genome sequences deposited in GenBank, twelve pairs of primers were designed to amplify thefragments spanning the entire genome of strain Duck/JS/10, and each fragment amplified by RT-PCRshared overlapping sequences with its neighbouring fragments. Through sequencing and editing,Duck/JS/10genome sequence was obtained. The genome organization of Duck/JS/10was identical tothose of other Class Ⅰ NDV strains, the genome size was15,198nts as expected. And the12-ntinsertion in the oding region of the P gene was also presented. Based on phylogenetic analysis withthe partial nucleotide sequence of F genes, Duck/JS/10was classified into the subgenotype4and thisis the first report of isolated genotype4virus in china.Both rabbit antiserum against JS10and La Sota presented cross HI and VN titers to differentNDV strains. HI titers of JS10antiserum to the tested virulent virus were3–5folds higher than thoseof La Sota antiserum. JS10antiserum showed more than10-fold higher inhibition than La Sotaantiserum to two class I NDV strains9a5b and JS10. VN assays of antisera were carried out in bothchicken embryos and DF-1cells. JS10antiserum showed higher VN titers to different NDV strainstested than La Sota antiserum, especially for class I viruses. Chickens in were injected with106EID50each of JS10or La Sota, respectively.Twenty one days later, chanllenged with class Ⅱ virulent strainZJ1. Obvious clinical signs or mortality can not observe in two immunized groups. And virusshedding from JS10vaccinated group is lower than in swabs from La Sota vaccinated group.Considering that i) JS10showed broader cross-antigenicity among NDV strains than current vaccinestrain La Sota; ii) JS10vaccinated chickens were better protected for virus shedding than thosevaccinated with La Sota; real potential of JS10as a therapeutic weapon for the treatment of ND inpoultry.To determine whether the entire avirulent virus have the potential to become highly pathogenic, wepassaged four benign NDV strains in chickens airsac and embryos respectively. During passage of theDuck/JS/10via air sacs, the pathogenicity value increased, reaching MDT, ICPI, and IVPI values of46, 1.94, and2.08, respectively. The F protein cleavage site has also mutant from112ERQERL117to112KRQKRF117. We compared the sequences of the Duck/JS/10and variant genomes (A10and E20). Atotal shared11mutations were identified between them and parent virus. Infection experiment confirmthat Duck/JS/10after passaged by air sac acquire the much high proliferation capacity than tamed inembryos.Seven fragments were amplified and cloned into T-easy vector with the designed primers. The3'and5' genome of Duck/JS/10was direct cloned in to transcription vector TVT TR and namedTVT-35. The fragments, amplified with5pair inner primer were subcloned into T-easy vectorsequentially to construct the plasmid T-AA. The resultant plasmid was digested with Afl Ⅱ, and then thefragment of interest was ligated into plasmid TVT-35, digested with the same enzymes, to construct the plasmidTVT-Duck/JS/10which contained the full-length cDNA of NDV Duck/JS/10strain. Three helperplasmids were constructed by subcloning the fragments, amplified with the designed primers, into theexpression vector PCI-neo. The mini genome of9a5b inserted GFP-TGL, with Duck/JS/10threehelper plasmids, PCI-NP, PCI-P and PCI-L, were cotranfected into BSR-T7/5cell expressing T7RNApolymerase.48h later, Oberseve the transfected cell under fluorescence microscopy the greenfluorescence can be detected, and it is indicated that the helper plasmid can be used for rescue virus.Newcastle disease virus (NDV) can replicate in tumor cells and induce apoptosis in late stages ofinfection. However, the interaction between NDV and cells in early stages of infection is not wellunderstood. Here we report that, shortly after infection, NDV triggers the formation ofautophagosomes in U251glioma cells, as demonstrated by an increased number of double-membranevesicles, GFP-microtubule-associated protein1light chain3(GFP-LC3) dot formations, and elevatedproduction of LC3Ⅱ. Moreover, modulation of NDV-induced autophagy by rapamycin, chloroquine orsmall interfering RNAs targeting the genes critical for autophagosome formation (Atg5and Beclin-1)affects virus production, indicating that autophagy may be utilized by NDV to facilitate its ownproduction. Furthermore, the class ⅡI phosphatidylinositol3-kinase (PI3K/Beclin-1pathway plays arole in NDV-induced autophagy and virus production. Collectively, our data provide a uniqueexample of a paramyxovirus that uses autophagy to enhance its production.