Newcastle Disease Virus Virulence Research Articles

The haemagglutinin–neuraminidase protein of velogenic Newcastle disease virus enhances viral infection through NF-κB-mediated programmed cell death

The haemagglutinin–neuraminidase (HN) protein, a vital membrane glycoprotein, plays a pivotal role in the pathogenesis of Newcastle disease virus (NDV). Previously, we demonstrated that a mutation in the HN protein is essential for the enhanced virulence of JS/7/05/Ch, a velogenic variant NDV strain originating from the mesogenic vaccine strain Mukteswar. Here, we explored the effects of the HN protein during viral infection in vitro using three viruses: JS/7/05/Ch, Mukteswar, and an HN-replacement chimeric NDV, JS/MukHN. Through microscopic observation, CCK-8, and LDH release assays, we demonstrated that compared with Mukteswar and JS/MukHN, JS/7/05/Ch intensified the cellular damage and mortality attributed to the mutant HN protein. Furthermore, JS/7/05/Ch induced greater levels of apoptosis, as evidenced by the activation of caspase-3/8/9. Moreover, JS/7/05/Ch promoted autophagy, leading to increased autophagosome formation and autophagic flux. Subsequent pharmacological experiments revealed that inhibition of apoptosis and autophagy significantly impacted virus replication and cell viability in the JS/7/05/Ch-infected group, whereas less significant effects were observed in the other two infected groups. Notably, the mutant HN protein enhanced JS/7/05/Ch-induced apoptosis and autophagy by suppressing NF-κB activation, while it mitigated the effects of NF-κB on NDV infection. Overall, our study offers novel insights into the mechanisms underlying the increased virulence of NDV and serves as a reference for the development of vaccines.

Deciphering whole genome sequence of a Newcastle disease virus genotype VII.2 isolate from a commercial poultry farm in India

Here we report the isolation and complete genome sequence of virulent Newcastle disease virus (NDV) during the outbreak in 2023. NDV is isolated from the infected egg-laying vaccinated chickens of poultry farms located in Raipur district in Chhattisgarh, India. Next-generation sequencing (NGS) revealed the present NDV isolate has a genome length of 15,118 nucleotides (GenBank: OR185447). The phylogenetic tree illustrates the evolutionary relationship among NDV species and populations indicating the present NDV strain (NDV/Chicken/RPR/01/23) classified as genotype VII in class 2. NDV isolate exhibited 99.19 % and 97.53 % similarities in the nucleotide sequence in comparison with other strains of NDV/Owl/Guwahati/01/20 (GenBank: MZ546197.1) from India and chicken/Banjarmasin/010/10 (GenBank: HQ697254.1) from Indonesia. The present NDV genome is closely aligned with the NDV/Owl/Guwahati/01/20 strain and was clustered in genotype VII in class 2 suggesting possible spillover among the viral strains. The amino acid sequence at the protein Fusion protein (F) cleavage sequence 112RRQKR*F117 which is distinctive with other lentogenic vaccine NDV strain of La Sota and postulated as a major determinant of NDV virulence. Since there is limited information available on the exact cause of the abrupt death of poultry chickens located in Raipur district in Chhattisgarh, India, the present study aims to examine molecular sequencing of circulating NDV strains causing high mortality in this region. The neutralization epitopes of virulent NDV strains will expedite in development next next-generation vaccines to control circulating viral strains across the globe. The derived scientific information on genetic diversity, current taxonomic classification, and antigenic variations that exist within the genome of NDV will help in developing strain-specific vaccines and implementing strict measures.

Cellular vimentin regulates the infectivity of Newcastle disease virus through targeting of the HN protein

The haemagglutinin-neuraminidase (HN) protein plays a crucial role in the infectivity and virulence of Newcastle disease virus (NDV). In a previous study, the mutant HN protein was identified as a crucial virulence factor for the velogenic variant NDV strain JS/7/05/Ch, which evolved from the prototypic vaccine strain Mukteswar. Furthermore, macrophages are the main susceptible target cells of NDV. However, the possible involvement of cellular molecules in viral infectivity remains unclear. Herein, we elucidate the crucial role of vimentin, an intermediate filament protein, in regulating NDV infectivity through targeting of the HN protein. Using LC‒MS/MS mass spectrometry and coimmunoprecipitation assays, we identified vimentin as a host protein that differentially interacted with prototypic and mutant HN proteins. Further analysis revealed that the variant NDV strain induced more significant rearrangement of vimentin fibres compared to the prototypic NDV strain and showed an interdependence between vimentin rearrangement and virus replication. Notably, these mutual influences were pronounced in HD11 chicken macrophages. Moreover, vimentin was required for multiple infection processes of the variant NDV strain in HD11 cells, including viral internalization, fusion, and release, while it was not necessary for those of the prototypic NDV strain. Collectively, these findings underscore the pivotal role of vimentin in NDV infection through targeting of the HN protein, providing novel targets for antiviral treatment strategies for NDV.

Pathologic Mechanisms of the Newcastle Disease Virus.

Newcastle disease (ND) has been a consistent risk factor to the poultry industry worldwide. Its pathogen, Newcastle disease virus (NDV), is also a promising antitumor treatment candidate. The pathogenic mechanism has intrigued the great curiosity of researchers, and advances in the last two decades have been summarized in this paper. The NDV's pathogenic ability is highly related to the basic protein structure of the virus, which is described in the Introduction of this review. The overall clinical signs and recent findings pertaining to NDV-related lymph tissue damage are then described. Given the involvement of cytokines in the overall virulence of NDV, cytokines, particularly IL6 and IFN expressed during infection, are reviewed. On the other hand, the host also has its way of antagonizing the virus, which starts with the detection of the pathogen. Thus, advances in NDV's physiological cell mechanism and the subsequent IFN response, autophagy, and apoptosis are summarized to provide a whole picture of the NDV infection process.

Analysis of Amino Acid Changes in the Fusion Protein of Virulent Newcastle Disease Virus from Vaccinated Poultry in Nigerian Isolates.

The roles of fusion gene in the virulence of Newcastle disease virus are well established, but the extent of its variation among the XIV, XVII, and XVIII genotypes reported in Central Africa and West Africa has until recently been understudied. In this study, virulent Newcastle disease virus (vNDV) was isolated from dead chickens among vaccinated flocks between March and April 2020. Fusion (F) gene was sequenced and analysed for characterization and information about genetic changes. Many substitutions were observed along the region and some of their functions are yet to be determined. Results showed that all study isolates have virulent cleavage site sequence 112-RRRKR-116/F117 and clustered within genotype XIVb. Sequence analysis showed K78R mutation in the A2 antigenic epitope in all isolates and more along the F-gene which varied in some instances within the isolates. Mutation in this A2 antigenic epitope has been reported to induce escape mutation to monoclonal antibodies generated using the NDV LaSota strain. The range of percentage nucleotide and amino acid homology between the study isolates and commercially available vaccine strains is 81.14%-84.39% and 0.175-0.211, respectively. This report provides evidence of vNDV among vaccinated chicken flock and molecular information about circulating vNDV strains in Kano State, Nigeria, which is useful for the development of virus matched vaccines. Newcastle disease (ND) surveillance and molecular analysis of circulating strains in this region should be encouraged and reported. Furthermore, ND outbreaks or cases among vaccinated poultry presented to veterinary clinics should be reported to the state epidemiologist. Nucleotide sequences were assigned accession numbers OK491971-OK491977.

Amino Acid Mutations in Hemagglutinin-Neuraminidase Enhance the Virulence and Pathogenicity of the Genotype III Newcastle Disease Vaccine Strain After Intravenous Inoculation

Newcastle disease virus (NDV), the causative agent that generally causes severe disease in poultry, continues to mutate and has thus evolved into 21 genotypes. We previously isolated a velogenic genotype III NDV JS/7/05/Ch that evolved from the vaccine strain Mukteswar, accompanying by amino acid mutations in Hemagglutinin-Neuraminidase (HN). Here, we sought to investigate the role of the mutant HN protein in NDV virulence. The HN genes of Mukteswar and JS/7/05/Ch were replaced reciprocally via reverse genetics, yielding two recombinant viruses rJS/MHN and rMu/JHN, respectively. rMu/JHN, in which the endogenous HN protein was replaced with the HN protein of JS/7/05/Ch, had a higher intravenous pathogenicity index (IVPI) value in chickens. Moreover, dual aa mutations (A494D and E495K from JS/7/05/Ch-type HN) were introduced into the HN protein of Mukteswar to generate the recombinant virus rMukHN494+495JS. This virus showed an equivalent IVPI value to that of rJS/7/05/Ch (generated from parental JS/7/05/Ch via reverse genetics). In vitro and in vivo assays further showed that A494D and E495K in HN induced antigenic changes, a higher replication level and a more intense inflammatory response. Taken together, these findings indicate that aa mutations in HN are crucial for the virulence of the genotype III Newcastle disease (ND) vaccine strain after intravenous inoculation. Our study further highlights that close surveillance is needed to monitor the genetic variation of ND vaccine strains.

Point mutation revealed the resurgence of sub-genotype VII.2 Newcastle disease virus in Israel

ABSTRACT Real-time reverse transcription PCR (rRT-PCR) is a widely used method for Newcastle disease virus (NDV) diagnosis and classification. The virus matrix (M) gene is usually used for general NDV identification and the fusion (F) gene for virulence differentiation. This method is rapid and sensitive, but the high mutation rate of the viral genome is a source of concern, as it may cause diagnostic failure. In late 2019, we encountered a problem classifying virulent NDV in samples from a few poultry flocks in which clinical signs were observed. Although the general NDV identification test was positive, the rRT-PCR virulence differentiation test gave negative results or higher cycle threshold values (Ct) than expected. Partial F gene sequencing for unclassified clinical NDV samples was used to investigate the cause of the rRT-PCR virulence differentiation test failure. The cause of the diagnostic failure was found to be a mismatch at the 3′ end of the reverse primer and adjustments were made to the diagnostic assay. The partial sequencing also allowed us to differentiate the virus as virulent according to the amino acid sequence of the F gene cleavage site and to genetically classify the virus as sub-genotype VII.2 NDV. After realizing that this is a resurgence event of sub-genotype VII.2 virus in an already endemic area of sub-genotype VII.1, we traced the spread of the new virus for several months. In addition, whole-genome sequencing of two 2020 sub-genotype VII.2 isolates was performed. GenBank search yielded related sequences from Jordan (2018) and Pakistan (2015–2016). RESEARCH HIGHLIGHTS In 2019, there was a resurgence of NDV from sub-genotype VII.2 in Israel, in an already endemic area of sub-genotype VII.1. A mismatch at the 3′ end of the reverse primer caused a diagnostic failure of the NDV virulence differentiation rRT-PCR assay. The 2019 NDV sub-genotype VII.2 virus is genetically close to viruses from Jordan (2018) and Pakistan (2015–2016).

Chicken ISG12(2) attenuates Newcastle disease virus and enhances the efficiency of Newcastle disease vaccine via activating immune pathways.

Low virulence and strong immunogenicity are quite important for Newcastle disease virus (NDV) producing Newcastle disease (ND) living-attenuated vaccine. However, immunogenicity of NDV positively correlates to its virulence. Usually, the velogenic NDV induces stronger immune responses of poultry than the lentogenic strain, but virulent NDV poses a risk for chicken. In this study, we identified the chicken interferon (IFN)-stimulated gene 12-2 (ISG12(2)) not only attenuated NDV, but also increased immunogenicity of ND vaccine strain. First, we found that NDV infection or IFNs stimulation induced expression of chicken ISG12(2) that reinforced expression of IFNs. Over-expression or knock-down proved that chicken ISG12(2) inhibited NDV replication. Then, recombinant NDV LaSota strains (rLaSota/Fmut/ISG12(2) and rLaSota/ISG12(2)), expressing ISG12(2), were rescued. Pathogenicity tests showed that ISG12(2) expression attenuated NDV. RNA-seq or RT-qPCR demonstrated that, comparing to rLaSota/Fmut and rLaSota, rLaSota/Fmut/ISG12(2) and rLaSota/ISG12(2) induced hosts to produce cytokines enriching in innate and adaptive immune pathways in vitro and in vivo. Finally, we showed that rLaSota/ISG12(2) vaccination improved immune condition of chicken to quickly respond NDV infection and then enhance protection. These results suggest that chicken ISG12(2) is a potential novel molecular adjuvant to regulate immune responses, which decrease virulence and increase immunogenicity of NDV. The chicken ISG12(2) may contribute to the development of high efficient poultry vaccine.

The Influence of Newcastle Disease Virus Major Proteins on Virulence

The Newcastle disease virus (NDV) negative-strand RNA genome containssix genes. These genes encode nucleoprotein (NP), phosphoprotein (P),matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN),and RNA-dependent RNA polymerase (L) proteins. The six proteins affectthe virulence of NDV in different ways, but available information on thesix proteins is disparate and scattered across many databases and sources.A comprehensive overview of the proteins determining NDV virulence islacking. This review summarizes the virulence of NDV as a complex traitdetermined by these six different proteins.

Identification of a new amino acid mutation in the HN protein of NDV involved in pathogenicity

The fusion (F) and haemagglutinin-neuraminidase (HN) proteins of Newcastle disease virus (NDV) are viral entry proteins and are recognized as the major virulence determinants. Previously, a lentogenic NDV virus (CE16) was derived from a mesogenic strain (CI10) through sequential passages in chick embryos. Whole-genome sequence analysis revealed that the two homologous strains shared the same F protein but differed in HN with two amino acid (aa) substitutions (A215G and T430A). To elucidate the molecular reasons for virulence attenuation, two original plasmids (HN-CI10 and HN-CE16) and two single-point mutants (G215A and A430T) reverse-mutated from HN-CE16 were constructed to analyse the known biological functions of HN. The results showed that the A430T substitution significantly weakened the haemadsorption (HAd) activity, increased the neuraminidase (NA) activity, improved the fusion-promoting activity, and enhanced the cleavage-promoting activity of HN-CE16. However, G215A failed to induce obvious functional changes. Therefore, the aa residue HN430 may play a key role in determining virulence. To test this hypothesis, further studies on A430T were conducted through reverse genetics using an infectious cDNA clone. At the viral level, the A430T-mutated virus showed dramatic promotion of viral plaque formation, propagation, and pathogenicity in vitro and in vivo. This study demonstrates a new virulence site associated with HN protein functions, viral propagation, and pathogenicity. All these findings could lay a foundation for illuminating the molecular mechanism of NDV virulence.

Mutations in the Methyltransferase Motifs of L Protein Attenuate Newcastle Disease Virus by Regulating Viral Translation and Cell-to-Cell Spread.

ABSTRACTThe large (L) polymerase proteins of most nonsegmented, negative-stranded (NNS) RNA viruses have conserved methyltransferase motifs, (G)-G-G-D and K-D-K-E, which are important for the stabilization and translation of mRNA. However, the function of the (G)-G-G-D and K-D-K-E motifs in the NNS RNA virus Newcastle disease virus (NDV) remains unclear. We observed G-G-D and K-D-K-E motifs in all NDV genotypes. By using the infection cloning system of NDV rSG10 strain, recombinant NDVs with a single amino acid mutated to alanine in one motif (G-G-D or K-D-K-E) were rescued. The intracerebral pathogenicity index and mean death time assay results revealed that the G-G-D motif and K-D-K-E motif attenuate the virulence of NDV to various degrees. The replication, transcription, and translation levels of the K-D-K-E motif-mutant strains were significantly higher than those of wild-type virus owing to their altered regulation of the affinity between nucleocapsid protein and eukaryotic translation initiation factor 4E. When the infection dose was changed from a multiplicity of infection (MOI) of 10 to an MOI of 0.01, the cell-to-cell spread abilities of G-G-D- and K-D-K-E-mutant strains were reduced, according to plaque assay and dynamic indirect immunofluorescence assay results. Finally, we found that NDV strains with G-G-D or K-D-K-E motif mutations had less pathogenicity in 3-week-old specific-pathogen-free chickens than wild-type NDV. Therefore, these methyltransferase motifs can affect virulence by regulating the translation and cell-to-cell spread abilities of NDV. This work provides a feasible approach for generating vaccine candidates for viruses with methyltransferase motifs.IMPORTANCE Newcastle disease virus (NDV) is an important pathogen that is widespread globally. Research on its pathogenic mechanism is an important means of improving prevention and control efforts. Our study found that a deficiency in its methyltransferase motifs (G-G-D and K-D-K-E motifs) can attenuate NDV and revealed the molecular mechanism by which these motifs affect pathogenicity, which provides a new direction for the development of NDV vaccines. In addition to the (G)-G-G-D and K-D-K-E motifs of many nonsegmented, negative-stranded RNA viruses, similar motifs have been found in dengue virus, Zika virus, Japanese encephalitis virus (JEV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This suggests that such motifs may be present in more viruses. Our finding also provides a molecular basis for the discovery and functional study of (G)-G-G-D and K-D-K-E motifs of other viruses.

Hemagglutinin-neuraminidase Sequence and Phylogenetic Analysis of Two Newcastle Disease Virus Isolated from Chickens in Iran.

Newcastle disease is a highly contagious viral infection affecting many species of birds that can spread fast between poultry houses and cause a heavy economic burden on the poultry industry all around the world. Fusion and hemagglutinin-neuraminidase (HN) protein are important in the pathogenesis of the Newcastle disease virus (NDV). The HN protein is a critical viral protein with multiple functions and plays a key role in the formation of the virulence of NDV. Head of HN protein is responsible for receptor binding, neuraminidase activity. This study aimed to investigate the sequence homology of hemagglutinin-neuraminidase of two NDV isolates sampled from infected farms in Iran. The samples were collected from flocks that had been vaccinated by both types of live and killed vaccines for NDV. After isolation of NDV, the viruses were subjected to the polymerase chain reaction (PCR) amplification using two pairs of specific primers designed for the HN gene to amplify the complete HN gene (1730bp). Afterward, the PCR products were sequenced and analyzed by phylogenetic tree construction software. Based on the analysis, substantial sequence homology among Iranian isolates is within the range of 97.1-100%. Moreover, the sequence homology searching revealed a level of similarity between HN sequences of Iranian isolates and the HN sequences from other countries, particularly Asian ones. For instance, a high homology ratio (95.34%) was found between Iranian isolates and the sequences registered on online molecular databases from China. Based on phylogenetic analysis, the NDV isolates belong to the VIId genotype. Finally, it can be concluded that monitoring the circulation of NDVs among poultry and other birds can help to obtain an insight into the evolution of NDVs and control of panzootic viruses in the future.

Screening and mechanistic study of key sites of the hemagglutinin-neuraminidase protein related to the virulence of Newcastle disease virus

Newcastle disease is a kind of avian infectious disease caused by Newcastle disease virus (NDV). The virulence of NDV is dependent mainly on the fusion (F) protein and hemagglutinin-neuraminidase (HN) protein. The genomes of 2 viruses, NDV-Blackbird and NDV-Dove, are 99.9% similar, while NDV-Blackbird is a velogenic virus, and NDV-Dove is a lentogenic virus. Further analysis revealed that the F proteins of the 2 strains were identical, and only 5 amino acid sites on the HN proteins were inconsistent. Five different HN mutant plasmids were constructed and analyzed in this study. The results showed that the mutation F110L caused a significant increase in fusion-promotion activity caused by an increase in neuraminidase activity. Because of the increase in receptor-binding activity caused by G116R, there was a significant increase in fusion-promotion activity. The mutation G54S resulted in a slight decrease in the fusion-promotion activity caused by a slight decrease in receptor-binding activity. The slight increase in the fusion-promotion activity caused by A469V was associated with a significant increase in neuraminidase activity. Therefore, the amino acids L110 and R116 played a key role in determining the virulence difference between NDV-Blackbird and NDV-Dove, which could lay a foundation for illuminating the virulence differences of NDV strains, as well as the development of attenuated vaccines.

Identification of Newcastle disease virus P-gene editing using next-generation sequencing.

Avian paramyxoviruses 1 has the ability to edit its P gene to generate three amino-coterminal proteins (P, V and W), but its kinetic change is unclear. In this study, next-generation sequencing (NGS) was used to analyze the P-gene editing of Newcastle disease virus (NDV). Transcriptome analysis of chicken embryonic tissues and bursa of fabricius showed the P-gene editing frequencies were 45.46–52.70%. To investigate the rules of P-gene editing along time, the ratio of PVW was determined by PCR based deep sequencing at multiple time points in cells infected with velogenic and lentogenic strain respectively. The results confirmed similar editing frequencies with transcriptome data and the PVW ratios were stable along time among different NDVs, but had a greater V-gene transcript on velogenic strain infection (P<0.001), which were different from previous reports. Also, it was shown that the number of inserted G residues in P-derived transcripts was not limited to +9G, and +10G transcripts were identified. These results confirmed the NDV P-gene editing frequencies and provided a novel point of view on NDV P-gene editing with NDV virulence.

Phosphoprotein Contributes to the Thermostability of Newcastle Disease Virus.

Newcastle disease (ND), caused by Newcastle disease virus (NDV), is highly contagious and represents a major threat to the poultry industry. The thermostable vaccines are not insensitive to heat and ease of storage and transportation, but the mechanism of NDV thermostability remains unknown. The phosphoprotein (P), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and large polymerase protein (L) are associated with NDV virulence. The association between F, HN, or L and viral thermostability has been, respectively, studied in different reports. However, the effects of P on NDV thermostability have not been demonstrated. Here, we utilized an existing reverse genetics system in our laboratory, to generate chimeric viruses by exchanging the P protein between the thermostable NDV4-C strain and the thermolabile LaSota strain. Chimeric viruses were found to possess similar growth properties, passage stability, and virulence, as compared to those of these parental strains. Interestingly, the thermostability of the chimera with P derived from the thermolabile LaSota strain was reduced compared to that of the parental virus, and P of the thermostable NDV4-C strain enhanced chimeric virus thermostability. Our data demonstrate that P is an important factor for the thermostability of NDV and provides information regarding the molecular mechanism of NDV thermostability; moreover, these results suggest a theoretical basis for using the NDV4-C strain as a thermostable vaccine.

Enhanced Replication of Virulent Newcastle Disease Virus in Chicken Macrophages Is due to Polarized Activation of Cells by Inhibition of TLR7.

Newcastle disease (ND), caused by infections with virulent strains of Newcastle disease virus (NDV), is one of the most important infectious disease affecting wild, peridomestic, and domestic birds worldwide. Vaccines constructed from live, low-virulence (lentogenic) viruses are the most accepted prevention and control strategies for combating ND in poultry across the globe. Avian macrophages are one of the first cell lines of defense against microbial infection, responding to signals in the microenvironment. Although macrophages are considered to be one of the main target cells for NDV infection in vivo, very little is known about the ability of NDV to infect chicken macrophages, and virulence mechanisms of NDV as well as the polarized activation patterns of macrophages and correlation with viral infection and replication. In the present study, a cell culture model (chicken bone marrow macrophage cell line HD11) and three different virulence and genotypes of NDV (including class II virulent NA-1, class II lentogenic LaSota, and class I lentogenic F55) were used to solve the above underlying questions. Our data indicated that all three NDV strains had similar replication rates during the early stages of infection. Virulent NDV titers were shown to increase compared to the other lentogenic strains, and this growth was associated with a strong upregulation of both pro-inflammatory M1-like markers/cytokines and anti-inflammatory M2-like markers/cytokines in chicken macrophages. Virulent NDV was found to block toll-like receptor (TLR) 7 expression, inducing higher expression of type I interferons in chicken macrophages at the late stage of viral infection. Only virulent NDV replication can be inhibited by pretreatment with TLR7 ligand. Overall, this study demonstrated that virulent NDV activates a M1-/M2-like mixed polarized activation of chicken macrophages by inhibition of TLR7, resulting in enhanced replication compared to lentogenic viruses.

Cleavage site of Newcastle disease virus determines viral fitness in persistent infection cells

Newcastle disease, caused by infection with virulent strains of Newcastle disease virus (NDV), poses a risk for the poultry industry. The virulence of NDV is mainly determined by the cleavage site of F protein. Lentogenic NDV can become velogenic after passages in SPF chicken brain and air sac based on some strains isolated from water birds, because the proportion of virulent-related strains gradually increases. In contrast, this proportion remains unchanged if NDV is passaged via 10-day-old SPF chicken embryos. This information suggests that environmental conditions rather than mutation affect NDV fitness in quasispecies. However, it is unknown how the environment selects virulent-related strains from a viral population. In this study, velogenic and lentogenic NDV marked by green or red fluorescence were used to establish persistent infection (PI) in BHK-21 cells. Monitoring viruses by different methods, we found that, without competition, persistently infected cells harbored lentogenic and velogenic NDV strains similarly in terms of viral release, viral spread and the period of persistent viral infection. In contrast, under competitive co-infection, velogenic NDV became dominant in quasispecies from the fifth passage of PI cells, which resulted in the progressive disappearance of the lentogenic NDV strain. This domination was concomitant with a short-term reduction in the superinfection exclusion and supernatant interference in PI cells resulting in a velogenic virus rebound. We concluded that virulent-related F protein cleavage site facilitates the spread and replication of NDV in conditions under which cells do not secret trypsin-like proteases and do not inhibit free virus infection, resulting in a gradual increase in virulent strains in quasispecies with the number of passages.

The glutamic residue at position 402 in the C-terminus of Newcastle disease virus nucleoprotein is critical for the virus

The nucleocapsid proteins (NPs) of Newcastle disease virus (NDV) and other paramyxoviruses play an important functional role during genomic RNA replication. Our previous study showed that the NP-encoding gene significantly influenced viral replication. Here, we investigated the roles of certain amino acid residues in the NP C-terminus in viral replication and virulence. Results showed that the glutamic acid residue at position 402 (E402) in the C-terminus of the NP is critical for RNA synthesis in the NDV mini-genome system. Mutation of E402 resulted in larger viral plaques that appeared more quickly, and increased the virulence of NDV. Further study indicated that the mutant virus had increased RNA levels during the early stages of virus infection, but that RNA replication was inhibited at later time points. These findings increase our knowledge of viral replication and contribute to a more comprehensive understanding of the virulence factors associated with NDV.

Different Origins of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein Modulate the Replication Efficiency and Pathogenicity of the Virus.

To investigate the exact effects of different origins of Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein to the biological characteristics of the virus, we systematically studied the correlation between the HN protein and NDV virulence by exchanging the HN of velogenic or lentogenic NDV strains with the HN from other strains of different virulence. The results revealed that the rSG10 or rLaSota derivatives bearing the HN gene of other viruses exhibited decreased or increased hemadsorption (HAd), neuraminidase and fusion promotion activities. In vitro and in vivo tests further showed that changes in replication level, tissue tropism and virulence of the chimeric viruses were also consistent with these biological activities. These findings demonstrated that the balance among three biological activities caused variation in replication and pathogenicity of the virus, which was closely related to the origin of the HN protein. Our study highlights the importance of the HN glycoprotein in modulating the virulence of NDV and contributes to a more complete understanding of the virulence of NDV.

Roles of the Polymerase-Associated Protein Genes in Newcastle Disease Virus Virulence.

The virulence of Newcastle disease virus varies greatly and is determined by multiple genetic factors. In this study, we systematically evaluated the roles of the polymerase-associated (NP, P and L) protein genes in genotype VII NDV virulence after confirming the envelope-associated (F and HN) proteins contributed greatly to NDV virulence. The results revealed that the polymerase-associated protein genes individually had certain effect on virulence, while transfer of these three genes in combination significantly affected the chimeric virus virulence, especially when the L gene was involved. These results indicated that the L protein was a major contributor to NDV virulence when combined with the homologous NP and P proteins. We also investigated viral RNA synthesis using NDV minigenome systems to assess the interaction between the NP, P, and L proteins, which showed that the activity of the polymerase-associated proteins were directly related to viral RNA transcription and replication.