Genomic Characterization Of Newcastle Disease Virus Strains From Different Hosts And Their V Proteins Effect On Interferon B Production In DF-1Cell Lines

Newcastle disease （ND） is one of the most serious infectious diseases of birds andeconomically important poultry diseases. Newcastle disease virus （NDV）, a sole member ofthe avian paramyxovirus serotype1（APMV-1）, belongs to the genus Avulavirus within theParamyxoviridae family and is the causative agent of ND. The enveloped virus possesses anegative-sense, single-stranded RNA genome approximately15.2kb in length. There isconsiderable virulence diversity among different hosts. In the past few years, the infection andhost range of NDV is expanding constantly, which bringing new challenges for the preventionand control of ND. Interferon （IFN） is the first line of defense against virus infection. It hasbecome clear that most paramyxoviruses encode V proteins function as an alpha interferonantagonist and play important role in viral pathogenesis. Similar to other paramyxovirus, Vprotein is generated by an RNA-editing event that occurs during the transcription of the NDVP gene and it plays a key role in antagonism alpha interferon. However, NDV strains contain agroup of complex viruses with different hosts, diverse virulence and many genotypes, themolecular evolution characteristics of their V proteins and effect on host interferon remainsunclear.To illustrate the genome evolution characteristics and the relationship between viral Vproteins, viral pathogenesis and host specificity among NDV isolates from different hosts.Based on analysis of many NDV isolates from different hosts in our lab, we selected threerepresentative NDV strains, which includes two from crested ibis and one from pig to studythe biology, genomic characterization and genetic variation of Newcastle disease virus strainsfrom different hosts, and further a total of twenty-two NDV strains were also compared toanalyze the molecular evolution characteristics of their V proteins and effect on hostinterferon, which including the other hosts derived virus such as chicken, goose, pigeon, andwild birds etc. This work contains five different parts and the details are presented infollowing paragraphs.1. Genomic characterization of two Newcastle disease virus strains isolated from Crested Ibis （Nipponia nippon） in ChinaTissue samples were obtained from ND-like disease of the crested ibis population in theShaanxi Rare and Wildlife Rescuing and Breeding Centre in February2006and January2010.Two Newcastle disease virus （NDV） isolates were collected from sick crested ibises and theirpathogenic and phylogenetic were investigated. Complete genome sequence of the twoisolates were obtained by RT-PCR and genomic analysis were conducted using DNAStar andMEGA4.0software. The pathogenic results indicated that the Shaanxi06isolate wasvelogenic, whereas the Shaanxi10isolate was mesogenic. They shared the same virulent motif¹¹²R-R-Q-K-R-F¹¹⁷ at the F protein cleavage site. Genomic characterization results showedthat both isolates consist of15,192nt, and there were tiny difference between the Shaanxi06（GenBank: KC853019）, Shaanxi10（GenBank: KC853020） isolates and other NDV referencestrains in genome characteristics, protein sequence coding and non-coding region of thegenome, but the two isolates owned new characteristics. The phylogenetic analysis revealedthat both isolates were clustered with class II NDVs, with one in genotype VIId and another ina novel genotype （provisionally designated as VIi）. The two isolates shared high homologywith the strains isolated from poultry flocks in the same region from2006to2010. However,both crested ibis isolates were genetically distinct from the traditional vaccine strains La Sota,B1and clone30strains, Chinese standard challenged strain F48E9and these isolatesclustered in different groups.2. Genomic characterization of a Newcastle disease virus strain HX01isolated from sickpigs in ChinaThe genomic characterization of one swine NDV isolate, HX01, is reported. HX01wasisolated from sick pigs suffering from an influenza-like disease in a commercial pig farm inShaanxi Province in December2009. The pathogenic tests and phylogenetic analysis wereinvestigated. Complete genome sequence of the swine isolate HX01was obtained by RT-PCRand genomic analysis was conducted using DNAStar and MEGA4.0software. Thepathogenic tests indicated that the isolate was a lentogenic NDV strain. HX01possessed a¹¹²G-R-Q-G-R-L¹¹⁷ lentogenic motif at the F protein cleavage site. The complete genomicsequence of HX01isolate consists of15,186nt （GenBank accession number: JF795531）,similar to other NDV reference strains in genome characteristics and is highly similar with theLa Sota vaccine strain （99.5%） and other swine-origin NDVs at either genome or single-genelevel. The phylogenetic analysis of F gene also showed that HX01belonged to genotype II,and is most closely related to the traditional vaccine strain La Sota, B1and clone30strains.New characteristics were found in the HN gene of HX01. A single amino acid change S526Nwas found within region2of the antigenic epitope in HN. In addition, a unique A-T mutation was observed in the HN gene end sequence in HX01. This finding may play an important rolein further research on NDV evolution.3. Cloning, molecular characteristics and genetic analysis of V proteins of Newcastledisease virus strainsBased on the published sequences of NDV P gene on GenBank, the P genes of twentyNDV strains of different hosts, genotypes and various virulence and two other NDV referencestrains were cloned and sequences, the corresponding V gene were obtained by inserting onenon-template G residue at the conserved editing sites of P gene of different NDV strains. Thesequence homology analysis, comparison with other reference strains and phylogeneticanalysis were conducted by using DNAStar and MEGA4.0software. The results showed thatV genes of twenty-two different genotypes and virulence NDV strains isolated from differenthosts were cloned and sequenced. The nucleotide similarity ranged from80.4%to100.0%,while deduced amino acid similarity ranged from67.5%to100%. The homology of the twentyNDV strains with other reference strains was from68.3%to100.0%. The results also indicatedthat the homology of V gene was high among same genotypes while low homology amongdifferent genotypes. There existed great variation in V protein among the current prevalentgenotype VII strains, traditional vaccine strain and Chinese standard challenged strain F48E9,also great differences among sub-genotypes within genotype VI strains. V protein structureanalysis showed that N-terminal residues55-105and C-terminal residues135-172wasthe most variable region, and the N-terminal MATF/LTDAEI, KKG （residues132-134）,HRRE （residues177-180）, SISW （residues182-185） and zinc finger structure region ofhistidine and seven cysteine residues was highly conservative in all NDV strain, somestrains with exception. The results also indicated that the genetic variation of V proteinsof different NDV strains were consistent with their genotypes. The phylogenetic analysisdemonstrated that the phylogenetic trees based on whole nucleotide sequence of V geneand V protein was highly consistent with that based on the47nt to420nt in the F gene’scoding region. The results indicated that the non-structural V gene can be applied ascandidate target gene to genetic analysis and molecular epidemiological studies in NDV,also suggested that genetic evolution of NDV is synchronous among all genes （includingstructural genes and nonstructural gene）.4. Differential interferon β production suppressing capacities of the V proteins fromdifferent Newcastle disease virus strains in DF-1cell linesTo study differential interferon β production suppressing capacities of the V proteinsfrom different Newcastle disease virus strains, seven NDV strains including different hosts,virulence and the genotypes were used to construct V protein eukaryotic expression vector pCMV-3HA-V. Reporter gene assay, RT-PCR and ELISA assays were applied to detectinterferon β in DF-1cells at different levels post transfection with various V constructs. Theresults showed that overexpression of V protein from different NDV strains were able tosuppress the IFN-β promoter activity, mRNA and protein levels in response to poly （I:C） inDF-1cells. The expression level of different V proteins was comparable to each other in DF-1cells, but their abilities to suppress IFN-β were different. The interferon β productionsuppressing capacities were from strong to weak in this order, pV-F48E9、pV-Sd-08、pV-Shaanxi06、pV-Gfw-10、pV-Shaanxi10and pV-HX01. The results indicated that the Vprotein of F48E9strain was most powerful in IFN-β antagonism, HX01is the most weakest,and the V proteins of other NDV strains were intermediate between them. It seems the Vproteins of velogenic strains were much stronger than that of mesogenic and lentogenicstrains in IFN-β suppressing capacities. The more virulent in virulence, more stronger ininhibiting IFN-β. It suggested that the V protein of NDV is closely related to the viralpathogenesis, but no correlation with the source of virus.5. Molecular determinants of V protein of Newcastle disease virus for interferon βproduction suppressing capabilityIn order to explore molecular determinants of V protein of Newcastle disease virus forinterferon β production suppressing capability and role of seven cysteine residues sited at theC-terminal cysteine-rich region of the NDV V protein. P protein, N-or C-terminal truncationsand seven cysteine mutants C1-C7were constructed and subcloned into plasmid pCMV-3HA.Western blotting, reporter gene assay, ELISA and real-time PCR were applied to detectinterferon β in DF-1cells at different levels post transfection with wide-type or mutant formsof NDV V proteins. Finally the zinc finger region of NDV V protein was modeled by usingPIV5V protein as a model, the results showed that the mutants were successful constructed,and high expression in DF-1cells. Overexpression of wide-type V protein of NDV were ableto suppress the IFN-β promoter activity, mRNA and protein levels in response to poly （I:C） inDF-1cells, but P protein, which shared the same N-terminal had no effect on suppressingIFN-β capability. The domain of NDV V protein was located at the C-terminal. Mutation ofcysteine196（C1）,221（C6）, or224（C7）, resulted in V proteins that retained the ability toinhibit IFN-β, indicating that these amino acids are not required for NDV V protein onsuppressing IFN-β capability. In contrast, mutation of cysteine200（C2）,213（C3）,214（C4）,or217（C5） resulted in V proteins defective in their ability to inhibit IFN-β, indicating thatthese residues are essential for NDV V protein on suppressing IFN-β capability. The resultsindicated that differential cysteine requirement observed with the NDV V protein. Schematicdiagram of the NDV V protein zinc finger structure showed that these residues of the smaller finger loops L1（C1, C6and C7） are not required for NDV V protein on suppressing IFN-βcapability while and these residues of the larger finger loops L2（C2, C3, C4and C5） wereessential.In summary, we firstly investigated and genomic characterization of the NDV strainsisolated from crested ibises and fully genomic analysis the swine NDV isolates with otherNDV strains. The genomic characterization of NDV strains of different hosts were useful toillustrate genome evolution, characteristics and genetic variation of NDV, also be helpful toclarify the virus origins, epidemic characteristics and molecular mechanism of the extensionof the host range. The study on molecular evolution characteristics and function on interferonβ production suppressing capacities of the V proteins from different Newcastle disease virusstrains can help us understand the role of NDV V protein in viral pathogenesis and theextension of the host range. The study reveal the relationship between the NDV V protein andviral pathogenesis, and it useful for further exploring the mechanisms of virus escapinginterferon, the function domain of NDV V protein on interferon antagonism, and long-termNewcastle disease vaccine develop.