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Growth kinetics of rescued wt and HN-mutated NDV viruses. The growth characteristics of viruses were determined by means of single and multi-cycle growth curves in both BHK-21F cells (a) and DF1 cells (b) infected with viruses at MOIs of 5 and 0.01, respectively. Supernatants were collected at 8-h or 12-h intervals. Viral titers were quantitated in SPF embryonated chicken eggs. The data shown represent three independent experiments; bars represent the mean ± SD of three independent experiments (n = 3) (*P < 0.05, ***P < 0.001).
Source publication
Among the proteins encoded by Newcastle disease virus (NDV), the attachment protein (HN) is an important determinant of virulence and pathogenicity. HN has been molecularly characterized at the protein level; however, the relationship between the molecular character of HN and the animal pathotype it causes has not been well explored. Here, we revis...
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... of BHK-21F and DF1 cells. BHK-21F cells were infected with rG7-wt, rG7-P93A, or rG7-L94A at a multiplicity of infection (MOI) of 5 or 0.01. When inoculated at an MOI of 5, rG7-wt reached a titer peak at 32 h post-infection (hpi), rG7-P93A grew more slowly but for longer than rG7-wt, and its titer was higher than that of rG7-wt at 48 hpi (Fig. 4). However, at an MOI of 0.01, the titers of rG7-wt and rG7-P93A continued to increase until the end of the observation period (72 hpi). The rG7-L94A virus was markedly attenuated, with a consistently low growth velocity in BHK-21F cells at 5 and 0.01 MOI. To confirm the growth of these viruses in avian cells, DF1 cells were infected ...
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... the end of the observation period (72 hpi). The rG7-L94A virus was markedly attenuated, with a consistently low growth velocity in BHK-21F cells at 5 and 0.01 MOI. To confirm the growth of these viruses in avian cells, DF1 cells were infected with the three rescued viruses. The growth kinetics of three viruses was similar in BHK-21F and DF1 cells (Fig. 4b). These results indicate that the P93A mutation barely influences the growth of NDV in cells, but the L94A mutation severely disrupts ...
Context 3
... functions. The fusogenic abilities of the rG7-P93A and rG7-L94A viruses were approximately 70% of that of rG7-wt. Phenotypically, fewer syncytia were induced by rG7-P93A than by rG7-wt, whereas smaller syncytia were formed in the cells infected with rG7-L94A (Fig. 5b), which resulted in an earlier cytopathic effect and lower viral replication (Fig. 4). The P93A and L94A substitutions attenuate the replication and pathogenicity of G7 in specific pathogen-free chickens. To understand whether the different biological phenotypes of rG7-P93A and rG7-L94A in vitro correlate with pathotypes in embryonated chicken eggs or specific pathogen-free (SPF) chickens in vivo, we conducted three ...
Similar publications
Newcastle disease (ND) is one of the most serious diseases affecting numerous avian species worldwide. However, data on the genetic characterization of ND virus (NDV) in the Philippines are limited. Herein, the complete sequences of the fusion (F) and hemagglutinin-neuraminidase (HN) genes of a NDV strain isolated in Central Luzon in 2017 were gene...
Citations
... The viral RNA genome encodes six major structural proteins: nucleocapsid (N), matrix (M), phosphoprotein (P), fusion (F), haemagglutinin-neuraminidase (HN) and large polymerase (L), of which only the F and HN glycoproteins interact with the viral envelope and are significant contributors to pathogenic and antigenic features, as well as key players in determining the NDV strain host range (Cho et al., 2007;Huang et al., 2004;Yan et al., 2020). Moreover, it demonstrated that these two glycoproteins on the external surface of the viral envelope elicit neutralizing antibodies to protect hosts against NDV (Collins et al., 1993;Liu et al., 2015;Sabouri et al., 2018). ...
... HN is another critical virulence factor of NDV. It acts as a multifunctional surface glycoprotein and mediates various activities, such as interacting with the F protein to promote membrane fusion and virus penetration (Estevez et al., 2011;Huang et al., 2004;Khattar et al., 2009;Liu et al., 2015). In addition, haemagglutination assay (HA) causes erythrocyte aggregation and attachment to sialic acid-containing receptor(s) on cell surfaces as well as actings on neuraminidase (NA) to release sialic acid from progeny virus particles, preventing viral self-aggregation. ...
... Based on the previously published reports (Yan et al., 2020;Sabouri et al., 2018;Liu et al., 2015;Ke et al., 2010;Zaitsev et al., 2004;Li et al., 2015), the residues in positions 401, 416 and 526 are determined as receptor binding sites, and residues from 234 to 239 are known as sialic acid binding sites. For these two regions, there were no observed substitutions among different genotypes (Table 3) which is known as the primary linear epitope of HN (Cho et al., 2007;Cho et al., 2008). ...
Background
Haemagglutinin–neuraminidase (HN) is one of the membrane proteins of Newcastle disease virus (NDV) that plays a significant role during host viral infection. Therefore, antibodies against HN are vital for the host's ability to protect itself against NDV infection due to their critical functions in viral infection. As a result, HN has been a candidate protein in vaccine development against the Newcastle disease virus.
Methods
This report used the full‐length sequence of the HN protein of NDV isolated in Iran (VIId subgenotype). We characterize and identify amino acid substitutions in comparison to other more prevalent NDV genotypes, VII subgenotypes and vaccine strains. Furthermore, bioinformatics tools were applied to determine the three‐dimensional structure, molecular dynamics simulation and prediction of B‐cell antigenic epitopes.
Results
The results showed that the antigenic regions of our isolate are quite comparable to the other VII subgenotypes of NDV isolated from different geographical places. Moreover, by employing the final 3D structure of our HN protein, the amino acid residues are proposed as a B‐cell epitope by epitope prediction servers, which leads to the introduction of linear and conformational antigenic sites.
Conclusions
Immunoinformatic vaccine design principles currently exhibit tremendous potential for developing a new generation of candidate vaccines quickly and economically to eradicate infectious viruses, including the NDV. In order to accomplish this, focus is directed on residues that might be considered antigenic.
... Its genome has six open reading frames (ORF), which encode six major structural proteins, namely, nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutininneuraminidase (HN), and the RNA-dependent RNA polymerase (L) in the order 3 ′ -NP-P-M-F-HN-L-5 ′ , and two non-structural proteins (V and W) generated by the P-gene mRNA editing [7,8]. The F and HN proteins are the main virulence factors of the virus [9,10] and play essential roles in the assembly and development of enveloped viruses, determining tropism in the host and tissues and facilitating the attachment of viruses to the host cell [11]. ...
Poultry production is essential to the economy and livelihood of many rural Zambian households. However, the industry is threatened by infectious diseases, particularly Newcastle disease virus (NDV) infection. Therefore, this study employed next-generation sequencing to characterise six NDV isolates from poultry in Zambia’s live bird markets (LBMs) and wild waterfowl. Four NDV isolates were detected from 410 faecal samples collected from chickens in LBMs in Lusaka and two from 2851 wild birds from Lochinvar National Park. Phylogenetic analysis revealed that the four NDVs from LBM clustered in genotype VII and sub-genotype VII.2 were closely related to viruses previously isolated in Zambia and other Southern African countries, suggesting possible local and regional transboundary circulation of the virus. In contrast, the two isolates from wild birds belonged to class I viruses, genotype 1, and were closely related to isolates from Europe and Asia, suggesting the possible introduction of these viruses from Eurasia, likely through wild bird migration. The fusion gene cleavage site motif for all LBM-associated isolates was 112RRQKR|F117, indicating that the viruses are virulent, while the isolates from wild waterfowl had the typical 112ERQER|L117 avirulent motif. This study demonstrates the circulation of virulent NDV strains in LBMs and has, for the first time, characterised NDV from wild birds in Zambia. The study further provides the first whole genomes of NDV sub-genotype VII.2 and genotype 1 from Zambia and stresses the importance of surveillance and molecular analysis for monitoring the circulation of NDV genotypes and viral evolution.
... n eukaryotic and prokaryotic hosts and used as a vaccine in chickens, which induces high antibody titres in both expression systems (Shafaati et al. 2022b). The haemagglutinin neuraminidase (HN) and fusion (F) proteins are the main virulence components of the virus and are crucial for immunogenicity against it (Chen et al. 2021;Khattar et al. 2009;B. Liu et al. 2015;Yan et al. 2020). Two glycoproteins, F and HN, are crucial for the growth and assembly of enveloped viruses as well as for defining the tropism of the host and tissues (Huang et al. 2004;Yan et al. 2020). The HN protein is in charge of binding, while the F protein causes fusion (Gimenez et al. 2019;T. Liu et al. 2019;Ruan et al. 2020). H ...
... Viral replication, transcription, translation and protein processing all take place in the cytoplasm of the host cell, while virus particles are released through the plasma membrane through budding [8][9][10]. Haemagglutinin-neuraminidase (HN) and fusion (F) proteins play an essential role in immunogenicity against the virus and are the virus' key virulence factors [11][12][13][14]. Two glycoproteins, F and HN, play essential roles in the assembly and development of envelop viruses and determining tropism in the host and tissues [14,15]. ...
Newcastle disease virus (NDV) belongs to the genus Avaluvirus and
Paramyxoviridae family, and it can cause acute, highly contagious Newcastle disease
in poultry. The two proteins, HN and F, are the main virulence factor of the virus and
play an essential role in immunogenicity against the virus. In most paramyxoviruses,
the F protein requires HN protein to fuse the membrane, and HN proteins substantially
enhance the viruses fusion activity. The present study describes the successful cloning
of a Hemagglutinin–Neuraminidase protein from Newcastle Disease Virus and its
expression in B. subtilis WB800 using the modified shuttle vector pHT43.HNcoding
sequence was cloned into pGet II vector. It was then subcloned into shuttle vector
PHT43 and transferred to E.coli for replication. The recombinant plasmid was
extracted from E.coli and used to transform B. subtilis by electroporation. After
induction of recombinant B. subtilis by IPTG, total cell protein and the protein
secreted into media were analysed through a time course using SDS-PAGE. The
expressed HN protein was purified through cation-exchange chromatography followed
by metal affinity chromatography, using the 6×His epitope introduced at the carboxyl
terminus of the recombinant protein.The accuracy of the PHT43-HN construct was
confirmed by sequencing and enzymatic digestion analysis. SDS-PAGE results
showed that the recombinant HN protein was successfully expressed and secreted into
the medium. Moreover, the purified HN protein showed neuraminidase activity with
characteristics similar to the indigenous HN NDV protein. B. subtilis is a free
endotoxin host that could be a favourite prokaryotic platform for producing the
recombinant HN protein. The HN protein produced here could be considered and
evaluated as a candidate vaccine whose immunogenicity potential needs to be
assessed in animal models and proper control groups.
... The role of the highly conserved amino acids in the head region of the HN protein is critical, and changing various amino acids in this region disables the F-promoting ability of the HN protein and inhibits syncytia formation. An analysis of HN globular head structure shows that the NA activity site is associated with a β-sheet propeller motif [37] . The binding of the HN stalk region to sialic acid promotes an F protein conformational change and activates the fusion of the viral envelope with cell membranes [38] . ...
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.
... Correlations between pathogenicity and HN biological activities have been observed in some reverse genetics studies upon mutating specific residues [14][15][16]. P93Aand L94A-bearing viruses display impaired receptor recognition ability, NA activity, and fusion-promoting activity, all of which lead to virus attenuation. In addition, an L94A-mutated virus shows a dramatic decline in replication [14]. ...
... P93Aand L94A-bearing viruses display impaired receptor recognition ability, NA activity, and fusion-promoting activity, all of which lead to virus attenuation. In addition, an L94A-mutated virus shows a dramatic decline in replication [14]. The mutation Y526Q results in a decrease in viral HA activity, NA activity, and fusion activity and has an attenuating effect on growth kinetics in cell culture and pathogenicity [15]. ...
... Interestingly, the results also revealed some correlations between HN functions and viral replication in cells and chickens, which has also been reported previously [14,23]. Analyses of growth kinetics in cells and viral titres in tissue samples of chickens showed that rCE16-HNA430T had higher levels of viral replication than rCE16. ...
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.
... The two proteins, haemagglutinin-neuraminidase (NA; HN) and F, are the main virulence factor of the virus (B. Liu et al., 2015;Ren et al., 2019). Two glycoproteins F and HN play essential roles in the assembly and development of envelop viruses and determining tropism in the host and tissues (Jin et al., 2017). ...
Background
Newcastle disease (ND) virus (NDV) is one of the major pathogens in poultry farms that causes severe economic damages to the poultry industry, especially broiler chicken and turkey farms. Despite the endemicity of ND and its many epidemics in the country, the nature of the Iranian strain of the Newcastle virus is still largely unknown. This study aimed to characterise and evaluate NDV isolates obtained from commercial poultry farms in Iran in 2019 through haemagglutinin‐neuraminidase (HN) gene sequencing.
Method
HN gene of each NDV isolate was amplified and sequenced using specific primers followed by phylogenetic analysis of full length of HN gene open reading frame and amino acid (aa) sequence of HN.
Results
Phylogenetic analysis of the HN gene showed that the virus is very closely related to genotypes VII and III. Analysis of HN gene nucleotide sequences showed that all isolates encode proteins with a length of 571 aa.
Conclusion
Results of the present study are useful for a better understanding of molecular epidemiology of indigenous NDV strains and determining important molecular differences between fields and commonly used vaccine strains related to main immunogenic proteins.
... Additionally, two other proteins, V and W, are produced by RNA editing during transcription of the P gene (Motz et al., 2013). HN is an important immunogenicity protein and virulence factor for NDV (Huang et al., 2004;Khattar et al., 2009;Liu et al., 2015a). And the F protein cleavage site is a major determinant of NDV virulence (Connaris et al., 2002;Connolly et al., 2009). ...
... In the present study, we developed an antigen-matched genotype VII NDV vaccine candidate (G7M) by introduced mutations into the L and F genes of the virulent genotype VII G7 strain (Liu et al., 2015) using reverse genetics technology. The vaccine safety and genetic stability and the induced immune responses and protection against genotype VII NDV challenge were evaluated in chickens. ...
... BHK-21F cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies, CA, USA). The viral stock of NDV G7 strain was prepared as described previously (Liu et al., 2015). The vaccinia virus expressing T7 RNA polymerase (VVT7) was kindly provided by Dr. Zhigao Bu as a gift (Harbin Veterinary Research Institute, CAAS). ...
... The virulence of the recovered viruses was determined by the mean death time (MDT), the intracerebral pathogenicity index (ICPI) and the intravenous pathogenicity index (IVPI) as described previously (Liu et al., 2015). Genetic stability of the G7M virus was assessed by serially passaging in 9-day-old SPF hen embryos for 15 times and SPF chickens for 5 times. ...
... In our previous study on the role of the HN of G7 strain in the pathogenicity in chickens (Liu et al., 2015), we rescued a G7 mutant with highly increased fusogenic activity. Sequence analysis of the mutant identified two amino acid mutations (T458D and G459D) in the HRB linker domain of the F protein. ...
... To explore if the increased fusogenicity resulted from the F mutations affects other biological properties of the virus, we generated two NDV recombinant viruses bearing T458D or G459D mutation (designed as rG7-T458D and rG7-G459D, respectively) based on the rG7 infectious clone (Liu et al., 2015). First, we examined the capability of the attachment and replication of the mutated NDVs in BHK-21 cells at 0 h and 12 h post-infection by measuring the NP mRNA. ...
... BHK-21F, Vero and DF-1 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies, CA, USA). The viral stock of NDV G7 strain was prepared as described previously (Liu et al., 2015). The vaccinia virus expressing T7 RNA polymerase (VVT7) was kindly provided by Dr. Zhigao Bu as a gift (Harbin Veterinary Research Institute, CAAS). ...
The fusion (F) protein of Newcastle disease virus (NDV) affects viral infection and pathogenicity through mediating membrane fusion. Previously, we found NDV with increased fusogenic activity in which contained T458D or G459D mutation in the F protein. Here, we investigated the effects of these two mutations on viral infection, fusogenicity and pathogenicity. Syncytium formation assays indicated that T458D or G459D increased the F protein cleavage activity and enhanced cell fusion with or without the presence of HN protein. The T458D- or G459D-mutated NDV resulted in a decrease in virus replication or release from cells. The animal study showed that the pathogenicity of the mutated NDVs was attenuated in chickens. These results indicate that these two single mutations in F altered or diminished the requirement of HN for promoting membrane fusion. The increased fusogenic activity may disrupt the cellular machinery and consequently decrease the virus replication and pathogenicity in chickens.
