Phylogenic Analysis Of Newcastle Disease Viruses Isolated In Eastern China During 2005-2008 And Comparative Proteome Analysis Of Cells Infected With The Representative Viruses

Newcastle disease (ND) is an acute and highly contagious avian disease with worldwide distribution that can cause substantial economic losses. The causative agent, Newcastle disease virus (NDV), is a member of the genus Avulavirus in the family Paramyxoviridae. The genome of NDV is a non-segmented, single-stranded, negative-sense RNA. The NDV genome contains six genes (in the following order: 3′-NP-P-M-F-HN-L-5′) which code for nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN) and an RNA dependent RNA polymerase (L), respectively. F and HN are the two important membrane glycoproteins which directly determine the virus virulence and pathogenicity. To elucidate the molecular epidemiology of recent NDV isolates, seventy-nine Newcastle disease viruses (NDV) isolated from clinical specimens of different poultry species including chickens, pigeons, geese and ostriches in Eastern China during 2005–2008 were characterized biologically and phylogenetically. The results showed that these viruses represented diverse phylogenetic and pathogenic phenotypes, while, the subgenotypeⅦd NDV are still the dominant strains circulating in China. Based on the phylogenic analysis of NDV isolates, three NDVs with different genetic background were chosen to conduct the pathogenicity tests both in vivo and in vitro. The results displayed that subgenotypeⅦd NDV possessed high pathogenicity to chickens, geese and pigeons, while the subgenotypeⅥb NDV only exhibited host-specificity to pigeons. To further evaluate the difference in the pathogenicity induced by these three NDVs, the differential expression of proteins in the virus-infected cells was also analyzed via comparative proteome.1. Characterization of the NDVs isolated during 2005-2008Seventy-nine Newcastle disease viruses (NDVs) isolated from clinical specimens of different poultry species including chickens, pigeons, geese and ostriches in Eastern China during 2005–2008 were characterized biologically and phylogenetically. Both the F and HN gene sequences analysis showed that these isolates could be divided into two separate clusters: classes I (3) and II (76). Three class I viruses and one genotypeⅠand 12 genotypeⅡviruses of class II were avirulent, four genotypeⅥb viruses isolated from pigeons were moderately virulent, and two genotypeⅢviruses and 57 genotypeⅦd viruses were highly virulent. The sequence identity of genotype,ⅡandⅢwith field isolates vaccine strains Queensland V4, La Sota and Mukteswar were 99.6%, 96.2-99%, and 99.9% respectively, implying that they were derivatives of the corresponding vaccine strains widely used in Chinese poultry flocks. The four genotypeⅥb NDVs were all pigeon-origin, indicating the host-specific preference of viruses in this genotype. On the phylogenic tree, the 57 subgenotypeⅦd isolates were further divided into two subgroups,Ⅶd1 andⅦd2, in which most of the strains isolated in recent two years belonged toⅦd2. Compared ofⅦd1 viruses, the viruses inⅦd2 presented the four amino acid sequence mutations of N145K, Q279H, K387R and G520V in F protein and three mutations of T102I, A118E and T443M in HN, respectively. These NDV isolates exhibited the diversity both in phylogenesis and pathogenicity, and provided suitable biomaterials to fully investigate the pathogenesis of NDVs with different genetic background.2. Pathogenicity of NDVs with different genetic backgroundBased on the genetic and biological characteristics, three NDV isolates with different host origins and genotypes, including a goose isolate JS-5-05-Go (subgenotypeⅦd), a pigeon isolate WX-10-07-Pi (subgenotypeⅥb ) and a chicken isolate JS-7-05-Ch (genotypeⅢ), were seleted to study their pathogenicity. On the cellular level, chicken embryo fibroblast (CEF), duck embryo fibroblast (DEF) and goose embryo fibroblast (GEF) were infected with these viruses respectively at an MOI of 0.01. The results showed that the peak HA titers in culture supernatants induced by JS-5-05-Go were higher than that induced by the other two viruses. HA titers in culture supernatants of three avian embryo fibroblasts induced by WX-10-07-Pi were similar, whereas, those induced by JS-7-05-Ch were different. Additionally, GEF exhibited a slower disintegration process than DEF or CEF afterinoculation with the three viruses. To evaluate the pathogenicity of those NDVs with different genetic background in vivo, pigeons, specific-pathogen-free (SPF) chickens and geese were infected with the three viruses via natural route, with a virulent NDV strain F48E8 as a control virus. The results showed that only JS-5-05-Go and F48E8 could cause the death in the experimental pigeons. Pigeons inoculated with WX-10-07-Pi and JS-5-05-Go shed the virus intermittently, while pigeons infected with WX-10-07-Pi experienced much longer time in virus shedding and showed higher rate of virus isolation when compared with pigeons infected by other two strains. Therefore, genotypeⅥb NDV strain exhibited host-specifity to pigeons and could spread more easily in pigeons than other genotypes. The results in SPF chickens by natural route of infection showed that virulent strain JS-7-05-Ch was not highly pathogenic to chickens, whereas, the pathogenicity of the other three strains was closely related with their virulence. According to the extent of pathological changes, four viruses presented obvious variation in pathogenicity for geese and F48E8 demonstrated the highest pathogenicity, followed by JS-5-05-Go, JS-7-05-Ch and WX-10-07-Pi. Notably, no clinical sign was detected in geese infected with WX-10-07-Pi. Although F48E8 and JS-5-05-Go showed variation in pathogenicity for geese, no apparent differences in virus shedding and virus distribution in nine tissues were detected.3. Comparative proteome analysis of CEFs infected with NDVs of different genetic backgroundTo elucidate the interactions between host cells and NDVs with different genetic background on the cellular protein level, the proteomics technology of two-dimensional gel electrophoresis (2-DE) together with matrix associated laser dissociation / ionization time of flight mass spectrometry (MALDI-TOF-MS) was used to analyze proteomes of CEFs infected with viruses of different genetic background. Protein samples of CEFs were harvested at 24h and 48h postinoculation and prepared for 2-DE analysis. The results showed that 43 protein spots in total expressed differentially during virus infection, including persistent up-regulated protein spots, persistent down-regulated protein spots, up-regulated at antephase and down-regulated at anaphase protein spots and down-regulated at antephase and up-regulated at anaphase protein spots. Some proteins have differentially expressed levels in three NDV-infected groups. With MALDI-TOF-MS, 41 protein spots representing 39 proteins were successfully identified. Cellular functional protein analysis showed that these differentially expressed proteins related to viral protein (2%), cytoskeleton (41%), stress response (10%), protein translation and elongation (7%), RNA processing and biosynthesis (12%), energy metabolism-associated proteins (7%), signal transduction (12%), ubiquitin-proteasome pathway (2%)and other proteins (7%). The result of Western-blot analysis further demonstrated thatβ-actin andα-tubulin showed difference in expression during virus infection, confirming the results of 2-DE. Based on the protein functions, we concluded that in NDV infected CEFs, the up-regulated expression proteins, heat shock 27kDa protein 1 (HSP27), thioredoxin domain containing 5 (TXNDC5) and nucleophosmin 1 (NPM1) may inhibit or delay early apoptosis, and that the up-regulated expression of Elongation factor Tu, mitochondrial precursor (EF-Tu) and 40S ribosomal protein SA, (34/67 kDa laminin receptor) (PRSA) may be conducive to the synthesis of viral proteins. Furthermore, certain kinds of host defense mechanism might be developed to antagonize virus infection, such as the increased level of apoptosis induced by up-regulated heat shock protein 90kDa beta, member 1, (GRP94).Changes in the expression level of cellular microfilament-associated proteins beta-actin (ACTB) and Actin, cytoplasmic type 8 (ACTG8), signal transduction protein nucleolin (C23), small glutamine-rich tetratricopeptide (SGT) and 14-3-3 protein epsilon (14-3-3E) and translation and elongation protein TXNDC5 were related to the infection of WX-10-07-Pi. Alterations of the expression of cytoskeleton protein TUBA, keratin 19 (KRT19), plastin 3 (T isoform) (PLS3) and capping protein (actin filament) muscle Z-line, alpha 2 (CAPZA2), RNA processing protein heterogeneous nuclear ribonucleoprotein K (hnRNPK) and functionally unidentified protein similar to coiled-coil domain containing 46 (CCDC46) were associated with the infection of JS-7-05-Ch. Cytoskeleton protein vimentin (VIM) and Tubulin alpha-1 chain (TUBA1C), heat shock protein Grp94, protein translation and elongation EF-Tu and early endosome antigen 1 (EEA1) were considered the symbol proteins in the cells infected by JS-5-05-Go. Taken together, the data showed that cells could give arise to different responses to the infection of NDVs with different genetic background.Conclusions:(1) Seventy nine Newcastle disease viruses isolated during 2005-2008 were characterized, indicating diversify in phylogenesis and virulence. In China, genotypeⅦNDV was still the predominant genotype which could be divided into two subgroupsⅦd1 andⅦd2.(2) GenotypeⅠ,ⅡandⅢfiled isolates in this study shared high homology with vaccine stains V4, LaSota and Mukteswar, respectively, indicating that they were derivatives of the vaccines viruses. Compared with other genotype strains, subgenotypeⅥb NDV exhibited host-specificity to pigeons.(3) In this study, we first used the comparative proteome approach to investigate the differentially expressed proteins of chicken embryo fibroblasts infected by NDVs with different genetic backgrounds, and successfully identified 41 differentially expressed spots representing 39 proteins.(4) NDV infection induced a series of cellular responses, including alteration of cytoskeleton networks, disorders in the ubiquitin-proteasome pathway, disruption of the host translation mechanism, and inhibition of RNA processing and energy metabolism.(5) Symbolic proteins had been found which was related to the infected by the viruses with different genetic background.(6) The entry into cells of NDV is believed to occur by direct fusion at the plasma membrane. In addition, NDV may enter host cells by an endocytic pathway.