The Bioinformatics Analysis Of NDV And The Molecular Mechanism In Virulence Increase Of Muktswar Strain

Newcastle disease (ND) is one of the most devastating diseases in poultry. The causative agent, Newcastle disease virus (NDV), is a member of the Avulavirus genus in the Paramyxoviridae family. Although NDV has only one serotype, substantial antigenic and genetic diversity have been previously recognized. At least11genotypes (genotypes I to XI) have been described for NDVs. According to the latest proposed classification, NDV have been divided into16genotypes and more sub-genotypes. The main dynamics of evolution in nonsegmented RNA viruses is due to the inherent error rate of RNA replication associated with the RNA-dependent RNA polymerase. and some researchers consider recombination may play an important role in the evolution of NDV. While polymerase error is believed to be the main driving force for NDV evolution, it has been established that recombination in nonsegmented negative-sense RNA viruses, including NDV. is rare. In this study. A comprehensive dataset of NDV genome sequences was evaluated using bioinformatics to characterize the evolutionary forces affecting NDV genomes. Furthermore, NDV strains can be categorized according to their virulence into low (lentogenic), intermediate (mesogenic) or highly (velogenic) virulent strains. Base on the alignment of genomes, we found that two genotype III viruses, JS/7/05/Ch and Mukteswar possessing significant difference in virulence, shared more than99%identities in nucleotide sequence. The IVPI score of JS/7/05/Ch was remarkably higher than that of Mukteswar, even they shared the consistent cleavage site of virulent NDV strains. In order to elucidate the molecular mechanism responsible for the increased virulence of JS/7/05/Ch. the genomic difference between these two strains were thoroughly analyzed and identified. 1. Non-natural Recombination may influenced the evolutionary analysis of NDVDuring2005to2009. we characterized more than100NDV strains in our laboratory, but no recombinant strains were detected on the basis of the analysis of F and HN gene sequences. However, in recent years, more and more recombination events have been reported for NDVs. with the recombination occurring throughout the whole genome, even some recombinants of NDVs involved multiple genotypes have been reported. This controversy has prompted us to investigate whether the recombination events in NDVs are as common as has been reported. Eighty complete genomic sequences of NDV retrieved from GenBank (released before18Apr2011) were analyzed using RDP program. Our analysis indicated at least8strains were predicted to be recombinants in the80NDVs, including5strains generated from multi-recombination. Phylogenetic tree also confirmed the existence of recombination events in two available NDVs NDV/03/018and China/Guangxi09/2003. Using strains China/Guangxi09/2003and NDV/03/018as examples, we attempted to validate whether the unexpected high recombination rate was due to inaccuracy of some of the NDV sequences deposited in GenBank. The two viruses were plaque purified three times on primary chicken embryo fibroblasts and resequenced in this study. Surprisingly, the putative recombinations detected with the old versions of sequences in GenBank (DQ485230and GQ338310) did not exist with new seqences from purified sequences, indicating the artificial natue of the recombinations caused by polymerase template switching in these samples or laboratory-generated recombinants. To provide evidence for a potential non-natural recombinations resulting in a mosaic sequence, we performed reverse transcription-PCR (RT-PCR) amplification of the M gene from a mixed sample of different La Sota and ZJ1viruses, which represent genotypes â…¡ and â…¦. respectively. PCR products were cloned into pGEM-T vector (Promega). and multiple clones were sequenced. Clones carrying either genotype â…¡ or â…¦ sequences were identified. Notably, the two genotype sequences could also be identified in the same clone. These results suggest that non-natural recombination events can be induced with samples containing mixed virus genotypes or strains, probably through polymerase template switching during the PCR procedure. Collectively, some recombinant genome sequences in the GenBank are not nature-recombinations, which will influence the evolution analysis of NDV.2. The evolutionary dynamics of pigeon paramyxovirus-1Pigeon NDV. also named Pigeon paramyxovirus-1(PPMV-1). is an antigenic variant of avian paramyxovirus type1(APMV-1) of chickens that is responsible for an autonomous Newcastle disease (ND)-like entity in pigeons. During the1980s, this virus variants spread worldwide among racing and show pigeons. The F proteins of PPMV-1strains bear a poly-basic cleavage site motif characteristic of virulent NDV strains. However, most of the PPMV-1strains show low virulence based on the pathogenicity assays in chickens. Considering the unique feature of the PPMV-1. a comprehensive dataset of avian paramyxovirus-1(APMV-1) were evaluated using bioinformatics to characterize the evolutionary forces affecting PPMV-1genomes. Phylogenetic analysis of142full-length Newcastle disease virus genomes indicated that Pigeon paramyxo virus-1(PPMV-1) isolates all clustered to the same clade (sub-genotype VIb), suggesting that PPMV-1have a high host specificity to the pigeon (Columba livia). Phylogenetic analysis of731sequences of the complete coding region of F genes reinforced the suggestion that PPMV-1is host-specific to pigeons, while genotype â…¦d owns the widest range of host. According to the alignment of genome sequences without recombination,19specific amino acid residues were identified to differentiate PPMV-1from "classical" APMV-1. Matrix protein gene displayed higher yearly rates of change than the other five genes and the Time to Most Recent Common Ancestry (TMRCA) analysis showed that PPMV-1may appear early in1960s. The population history of PPMV-1displayed two major inflexion points, indicating its population size fluctuations. In1980s, the population size of PPMV-1underwent an explosive growth, whereas it showed a sudden decline in recent years. Additionally, compared to the other five genes, less positive selection sites were present in the HN gene, suggesting that HN gene suffered less immune pressure from the host. Result in this study showed that pigeon paramyxovirus-1is host-specific to pigeons and assumes a unique evolutionary history.3. Comparative analysis of the entire genomes of JS/7/05/Ch and MukteswarDuring the genome alignment, we found that two genotype III viruses, JS/7/05/Ch and Mukteswar showed significant difference in virulence while they shared more than99%identities in their genome sequences. The IVPI scores of the two cloned strains of JS/7/05/Ch JS-7122and JS-1217were2.2and2.15. while those of the two cloned strains of Mukteswar Muk-4and Muk-5were0.03and0.09, respectively. To elucidate the crtical genetic regions responsible for the incresed virulence of JS/7/05/Ch, the cloned strains JS-7122and Muk-4were selected for their entire genome sequence analysis. The alignment result showed that the main differences between these two NDV genomes were located in NP. HN and L genes while their P, M and F gene sequences were completely the same. There are3nucleotide mutations in NP gene with one site (1433nt) inducing the amino acid (AA) change and5mutations in L gene without involvement of AA substitutions. Notably, all the6mutations in HN gene caused the A A substitutions. The three-dimensional structures of HN protein of the two strains showed that there were4mutations on the surface of the protein. Furthermore, the AA substitutions at positions494and495are included in antigenic site12, which is also an important receptor-binding site of HN protein. Therefore, we surmised that HN gene may play a key role in the virulence increase of Mukteswar strain.4. The molecular mechanism in the virulence increase of Muktswar strainEight fragments from genomes of JS-7122and Muk-4were amplified and cloned into pCR2.1vector with the designed primers. The two fragments, NP and PM, were subcloned into pCR2.1vector sequentially to construct the plasmid pNPM. As the clone strategy mentioned above, the plasmid pMFHL composed fragments of MF, FH and HL while the plasmid pL123composed fragments L1, L2and L3. The plasmids pNPM and pL123were digested with Spe I and FspA I, and the targeted fragments from pNPM were purified and ligated into the pL123to construct the plasmid pNPML. The resultant plasmid was digested with Age I and FspA I, and then the fragment of interest was cloned into plasmid pMFHL, digested with the same enzymes, to construct the plasmid pALL which contained the full-length cDNA of NDV. Finally, the full-length cDNA was transferred to TVT7R(0.0) vector to construct full-length NDV infectious clone using specific enzymes. With the above clone steps, two infectious clones TVT/Muk-4, TVT/JS-7122were obtained. According to the genomic difference between the NDVs JS-7122and Muk-4, The HN genes of them were exchanged via molecular manipulations, resulting in two chimeric infectious clones Mu/JHN and JS/MHN, respectively. The four infectious clones (TVT/Muk-4, TVT/JS-7122, Mu/JHN and JS/MHN) with three helper plasmids, pCI-NP, pCI-P and pCI-L, were cotranfected into BSR-T7/5cell expressing T7RNA polymerase. After inoculation of transfected cell culture into embroynated chicken eggs from one specific pathogen free (SPF) flock, four NDV strains (rJS-7122, rMuk-4, rJS/MHN and rMu/JHN) were rescued successfully. The pathogenicity of the newly generated NDVs were characterized and the results showed that the virulence of rJS-7122and rMu/JHN were much higher than that of rMuk-4and rJS/MHN, suggesting that HN gene is the key factor for virulence increase of Mukteswar strain.