Newcastle disease virus (NDV) is a virus of paramyxovirus family and lately has been studied for the treatment of cancer in human. In this study, we successfully determined the oncolysis potential of NDV vaccine, V4UPM tested on the human glioblastoma multiform cell line (DBTRG.05MG) and human glioblastoma astrocytoma cell line (U-87MG) in vitro and in vivo. The V4UPM strain is a modified V4 strain developed as thermostable feed pellet vaccine for poultry.
1. Hubbard x Hubbard (HH) and Shaver x Shaver (SS) chicks given a dietary supplement of either 50 mg/kg oxytetracycline (OTC) or 1 g/kg Lactobacillus culture (LC) were exposed to 36 +/- 1 degrees C for 3 h daily from day (d) 21 to 42. 2. Prior to heat treatment, body weight (d 21) and weight gain (d 1 to d 21) of OTC and LC birds were greater than those fed the control diet. Chicks given LC had the best food efficiency followed by OTC and control birds during d 1 to d 21. Body weight (d 1 and d 21) and weight gain (d 1 to d 21) were greater for HH tlhan SS chicks. 3. After 3 weeks of heat exposure, birds receiving the LC diet had greater body weight and weight gain, higher food intake and lower food efficiency than OTC and control chicks. 4. Antibody production against Newcastle discase vaccine on d 21 was not affected by strain or diet. On d 42, while diet had negligible effect on this variable among the SS broilers, HH birds fed LC had higher antibody production than those on the control diet. 5. Neither strain nor diet had a significant effect on mortality.
Infectious bronchitis virus (IBV), an ongoing emergence enveloped virus with a single-stranded positive-sense RNA genome, belongs to the Gammacoronavirus genus in the Coronaviridae family. IBV-associated tracheitis, nephritis, salpingitis, proventriculitis and egg drop have caused devastating economic losses to poultry industry worldwide. Since the end of 2018, a remarkably increasing number of commercial broilers and layers, vaccinated or not, were infected with IBV in China. Here, we described two IB outbreaks with severe respiratory system or kidney injury in IBV-vaccinated commercial poultry farms in central China. Other possible causative viral pathogens, including avian influenza virus (AIV), Newcastle disease virus (NDV) and Kedah fatal kidney syndrome virus (KFKSV), were excluded by reverse transcription-polymerase chain reaction (RT-PCR), and three virulent IBV strains, HeN-1/China/2019, HeN-2/China/2019 and HeN-101/China/2019, were identified. Although the gross pathologic appearance of these two IB outbreaks was different, the newly identified IBV strains were all closely related to the ck/China/I0529/17 strain and grouped into GI-19 genotype clade based on the sequencing and phylogenetic analysis of the complete S1 genes. Moreover, there are still some evolutionary distance between the newly identified IBV strains, HeN-101/China/2019 in particular, and other GI-19 strains, suggesting that Chinese IBV strains constantly emerge and evolve towards different directions. In conclusion, this study provided an insight of the recently emerging IBV outbreaks in IBV-vaccinated commercial poultry farms and identified the genetic characteristics of three virulent GI-19 IBV strains, which shows the need to carry out proper preventive measures and control strategies.
The nucleotide sequence of the haemagglutinin-neuraminidase (HN) glycoprotein gene of Newcastle disease virus (NDV) variant strain V4(UPM) was determined by direct genomic RNA sequencing and confirmed by cycle sequencing. The gene comprises 1996 nucleotides encoding a 615 amino acid protein of size 67.4 kDa. The nucleotide and amino acid sequences of this strain were compared with those of the parent strain V4(QUE). There are 16 nucleotide substitutions on V4(UPM), eight of which are silent mutations and another eliminated a potential Asn-linked glycosylation site in V4(UPM). In addition, an Arg (403) residue was shown to be absent in the variant strain. This deletion is thought to be significant because of its location in a highly conserved region of the HN protein.
NP(Δc375) is a truncated version of the nucleocapsid protein of Newcastle disease virus (NDV) which self-assembles into a long helical structure. A packed bed anion exchange chromatography (PB-AEC), SepFastTM Supor Q pre-packed column, was used to purify NP(Δc375) from clarified feedstock. This PB-AEC column adsorbed 76.2% of NP(Δc375) from the clarified feedstock. About 67.5% of the adsorbed NP(Δc375) was successfully eluted from the column by applying 50 mM Tris-HCl elution buffer supplemented with 0.5 M NaCl at pH 7. Thus, a recovery yield of 51.4% with a purity of 76.7% which corresponds to a purification factor of 6.5 was achieved in this PB-AEC operation. Electron microscopic analysis revealed that the helical structure of the NP(Δc375) purified by SepFast(TM) Supor Q pre-packed column was as long as 490 nm and 22-24 nm in diameter. The antigenicity of the purified NP(Δc375) was confirmed by enzyme-linked immunosorbent assay.
Portions of the hemagglutinin neuraminidase (HN) gene of Newcastle disease virus (NDV) isolates from two recent outbreaks were sequenced to investigate epidemiology of this disease in Taiwan. These NDV isolates were all viscerotropic velogenic according to the clinical lesions produced in chickens. Sequence data were obtained from 14 NDV isolates (12 from 1995 and 2 from 1984). All isolates differed in their nucleotide sequences (from 0.3 to 15.3%), and represented potentially different strains of NDV. Phylogenetic analysis revealed that these isolates are closely related to viruses isolated from Japan and Malaysia. Some viruses isolated in 1995 appeared to evolve from viruses isolated in 1984. The results suggest that the 1995 outbreak of Newcastle disease (ND) in Taiwan may have been caused by multiple strains of velogenic NDV that have cocirculated in Taiwan for some time. Moreover, NDV isolates from racing pigeons were very similar to isolates from chickens in the same period, suggesting that both domestic and free-living birds were involved in the spread of ND in Taiwan.
Hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a vital role in the viral infectivity, host immunity, and disease diagnosis. A portion of the HN gene encoding the ectodomain (nt 142-1739) was cloned and expressed in Escherichia coli yielding an insoluble HN protein and a soluble NusA-HN protein containing N-utilization substance A (NusA) fusion component. Both recombinant proteins were purified and used for immunization of chickens. The recombinant HN protein induced higher antibody titers as compared to the recombinant NusA-HN protein. These antibodies were able to react in immunoblot analysis with the corresponding recombinant proteins as well as with the HN protein of NDV.
In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.
In the present work, a single-step purification of recombinant nucleocapsid protein (NP) of the Newcastle disease virus (NDV) directly from unclarified feedstock using an expanded bed adsorption chromatography (EBAC) was developed. Streamline 25 column (ID = 25 mm) was used as a contactor and Streamline chelating adsorbent immobilized with Ni2+ ion was used as affinity adsorbent. The dynamic binding capacity of Ni2+ -loaded Streamline chelating adsorbent for the NP protein in unclarified feedstock was found to be 2.94 mg ml(-1) adsorbent at a superficial velocity of 200 cm h(-1). The direct purification of NP protein from unclarified feedstock using expanded bed adsorption has resulted in a 31% adsorption and 9.6% recovery of NP protein. The purity of the NP protein recovered was about 70% and the volume of processing fluid was reduced by a factor of 10. The results of the present study show that the IMA-EBAC developed could be used to combine the clarification, concentration and initial purification steps into a single-step operation.
The nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV) viscerotropic-velogenic strain AF2240 was determined by direct RNA sequencing and by sequencing RT-PCR products. It encodes a single open reading frame of 581 amino acids with a calculated Mr of 63.8 kDa. The predicted sequence contains five asparagine glycosylation sites. Comparison of the AF2240 HN protein sequence with 13 other previously published sequences showed 88% homology. This HN protein is unique because it lacked the Arg 403 residue which is present in all of the other strains and cannot be grouped under the proposed three size classes of HN proteins in NDV.
Sequence analysis of the fusion (F) gene of eight Malaysian NDV isolates showed that all the isolates were categorized as velogenic viruses, with the F cleavage site motif (112)R-R-Q-K-R(116) or (112)R-R-R-K-R(116) at the C-terminus of the F(2) protein and phenylalanine (F) at residue 117 at the N-terminus of the F(1) protein. Phylogenetic analysis revealed that all of the isolates were grouped in two distinct clusters under sub-genotype VIId. The isolates were about 4.8-11.7% genetically distant from sub-genotypes VIIa, VIIb, VIIc and VIIe. When the nucleotide sequences of the eight Malaysian isolates were compared phylogenetically to those of the old published local isolates, it was found that genotype VIII, VII, II and I viruses exist in Malaysia and caused sporadic infections. It is suggested that genotype VII viruses were responsible for most of the outbreaks in recent years.
A two-step SYBR Green I real time polymerase chain reaction (PCR, real time PCR) for the detection of Newcastle disease virus (NDV) was developed. A melting curve analysis was performed to distinguish specific from non-specific products and primer dimers. Regardless of different virus pathotypes the melting temperature (Tm) ranged from 86 degrees C to 87 degrees C. The sensitivity of the real time PCR was compared with the reverse transcription (RT)-nested PCR enzyme-linked immunosorbent assay (ELISA, RT-nested PCR ELISA). Whereas the detection limit of the real time PCR was 10 pg DNA, the RT-nested PCR ELISA and conventional PCR could only detect up to 1 ng and 10 ng DNA, respectively. Thus the real time PCR offers a sensitive, rapid and convenient method for screening large number of NDV specimens.
SYBR Green I real-time PCR was developed for detection and differentiation of Newcastle disease virus (NDV). Primers based on the nucleocapsid (NP) gene were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. The assay was developed and tested with NDV strains which were characterized previously. The velogenic strains were detected only by using velogenic-specific primers with a threshold cycle (C(t)) 18.19+/-3.63 and a melting temperature (T(m)) 86.0+/-0.28 degrees C. All the lentogenic/vaccine strains, in contrast, were detected only when lentogenic-specific primers were used, with the C(t) value 14.70+/-2.32 and T(m) 87.4+/-0.21 degrees C. The assay had a dynamic detection range which spans over a 5log(10) concentration range, 10(9)-10(5) copies of DNA plasmid/reaction. The velogenic and lentogenic amplifications showed high PCR efficiency of 100% and 104%, respectively. The velogenic and lentogenic amplifications were highly reproducible with assay variability 0.45+/-0.31% and 1.30+/-0.65%, respectively. The SYBR Green I real-time PCR assay detected successfully the virus from tissue samples and oral swabs collected from the velogenic and lentogenic NDV experimental infection, respectively. In addition, the assay detected and differentiated accurately NDV pathotypes from suspected field samples where the results were in good agreement with both virus isolation and analysis of the fusion (F) cleavage site sequence. The assay offers an attractive alternative method for the diagnosis of NDV.
Newcastle disease (ND) is an extremely contagious and fatal viral disease causing huge economic losses to the poultry industry. Following recent ND outbreaks in Sabah in commercial poultry and backyard farms, it was speculated that this could be due to a new introduction of Newcastle Disease Virus (NDV) genotype/sub-genotype. Here we report the genetic characterization of NDVs isolated from Sabah during early 2021. All isolates were amplified and sequenced with primers specific to the viral fusion (F) gene using reverse transcription-polymerase chain reaction (RT-PCR). Nucleotide sequence analysis of the F gene showed that all isolates shared similar homology of 99.4% with NDV strain from Iran isolated in 2018. Amino acid sequences of the F protein cleavage site revealed the motif of 112RRQKRF117 indicating all isolates were of virulent strain. Phylogenetic analysis demonstrated that all isolates were clustered under sub-genotype VII 1.1 and clustered together with isolates from Iran (previously known as subgenotype VIIl). The present findings suggested that there is an emerging of a new sub-genotype into the poultry population in Sabah and this sub-genotype has never been reported before in Malaysia. Therefore, transboundary monitoring and continuous surveillance should be implemented for proper control and prevention of the disease. A further molecular epidemiological analysis of NDV is needed to well understand the circulatory patterns of virulent strains of NDV in the country to prevent future outbreaks.
One-day-old chickens were transported from Australia to Malaysia and vaccinated orotracheally with an uninactivated vaccine prepared from avirulent Australian V4 strain of Newcastle disease virus (NDV). The vaccination regimes were as follows: group A, once, at 2 weeks old; group B, once, at 3 weeks old; group C, twice, at 2 and at 3 weeks old; group D, direct contact with groups A, B, and C; and group E, indirect contact with groups A, B, C, and D. Group F was unvaccinated controls. Challenge was with NDV virulent Ipoh AF 2240-226 strain, administered at 4 weeks old intramuscularly to 10 chickens in each group and orotracheally to 10 chickens in each group. The remaining chickens were challenged by contact with the inoculated chickens. Group mortalities following challenge were: A, 1/77; B, 1/34; C, 0/39; D, 0/45; E, 6/43; and F, 60/60.
The complete VP1 protein of EV71 was truncated into six segments and fused to the C-terminal ends of full-length nucleocapsid protein (NPfl) and truncated NP (NPt; lacks 20% amino acid residues from its C-terminal end) of newcastle disease virus (NDV). Western blot analysis using anti-VP1 rabbit serum showed that the N-terminal region of the VP1 protein contains a major antigenic region. The recombinant proteins carrying the truncated VP1 protein, VP1(1-100), were expressed most efficiently in Escherichia coli as determined by Western blot analysis. Electron microscopic analysis of the purified recombinant protein, NPt-VP(1-100) revealed that it predominantly self-assembled into intact ring-like structures whereas NPfl-VP(1-100) recombinant proteins showed disrupted ring-like formations. Rabbits immunized with the purified NPt-VP(1-100) and NPfl-VP(1-100) exhibited a strong immune response against the complete VP1 protein. The antisera of these recombinant proteins also reacted positively with authentic enterovirus 71 and the closely related Coxsackievirus A16 when analyzed by an immunofluorescence assay suggesting their potential as immunological reagents for the detection of anti-enterovirus 71 antibodies in serum samples.
Newcastle disease virus (NDV) is an oncolytic virus which selectively replicates in cancer cells without harming normal cells. Autophagy is a cellular mechanism that breaks down unused cytoplasmic constituents into nutrients. In previous studies, autophagy enhanced NDV-induced oncolysis in lung cancer and glioma cells. However, the effect of autophagy inhibition on NDV-induced oncolysis in breast cancer cells remains unknown. This study aimed to examine the effect of autophagy inhibition on NDV-induced oncolysis in human breast cancer cells, MCF7. To inhibit autophagy, we knocked down the expression of the autophagy protein beclin-1 (BECN1) by short interfering RNA (siRNA). The cells were infected with the recombinant NDV strain AF2240 expressing green fluorescent protein. We found that NDV induced autophagy and knockdown of BECN1 significantly reduced the NDV-induced autophagy in MCF7 cells. Importantly, BECN1 knockdown significantly suppressed cell death by inhibiting viral replication, as observed at 24 h post infection. Overall, our data suggest that autophagy inhibition may not be a suitable strategy to enhance NDV oncolytic efficacy against breast cancer.
A Newcastle disease virus (NDV) isolate designated IBS002 was isolated from a commercial broiler farm in Malaysia. The virus was characterised as a virulent strain based on the multiple basic amino acid motif of the fusion (F) cleavage site (112)RRRKGF(117) and length of the C-terminus extension of the hemagglutinin-neuraminidase (HN) gene. Furthermore, IBS002 was classified as a velogenic NDV with mean death time (MDT) of 51.2 h and intracerebral pathogenicity index (ICPI) of 1.76. A genetic distance analysis based on the full-length F and HN genes showed that both velogenic viruses used in this study, genotype VII NDV isolate IBS002 and genotype VIII NDV isolate AF2240-I, had high genetic variations with genotype II LaSota vaccine. In this study, the protection efficacy of the recombinant genotype VII NDV inactivated vaccine was also evaluated when added to an existing commercial vaccination program against challenge with velogenic NDV IBS002 and NDV AF2240-I in commercial broilers. The results indicated that both LaSota and recombinant genotype VII vaccines offered full protection against challenge with AF2240-I. However, the LaSota vaccine only conferred partial protection against IBS002. In addition, significantly reduced viral shedding was observed in the recombinant genotype VII-vaccinated chickens compared to LaSota-vaccinated chickens.
Newcastle disease (ND) is a highly contagious avian disease and one of the major causes of economic losses in the poultry industry. The emergence of virulent NDV genotypes and repeated outbreaks of NDV in vaccinated chickens have raised the need for fundamental studies on the virus-host interactions. In this study, the profiles of B and T lymphocytes and macrophages and differential expression of 26 immune-related genes in the spleen of specific-pathogen-free (SPF) chickens, infected with either the velogenic genotype VII NDV strain IBS002 or the genotype VIII NDV strain AF2240, were evaluated. A significant reduction in T lymphocyte population and an increase in the infiltration of IgM+ B cells and KUL01+ macrophages were detected in the infected spleens at 1, 3 and 4 days post-infection (dpi) (P<0.05). The gene expression profiles showed an up-regulation of CCLi3, CXCLi1, CXCLi2 (IL-8), IFN-γ, IL-12α, IL-18, IL-1β, IL-6, iNOS, TLR7, MHCI, IL-17F and TNFSF13B (P<0.05). However, these two genotypes showed different cytokine expression patterns and viral load. IBS002 showed higher viral load than AF2240 in spleen at 3 and 4dpi and caused a more rapid up-regulation of CXCLi2, IFN-γ, IL-12α, IL-18, IL-1β, iNOS and IL-10 at 3dpi. Meanwhile, the expression levels of CCLI3, CXCLi1, IFN-γ, IL-12α, IL-1β and iNOS genes were significantly higher in AF2240 at 4dpi. In addition, the expression levels of IL-10 were significantly higher in the IBS002-infected chickens at 3 and 4dpi. Hence, infection with velogenic genotype VII and VIII NDV induced different viral load and production of cytokines and chemokines associated with inflammatory reactions.
A disulfide constrained random heptapeptide library displayed on filamentous bacteriophage M13 was applied to select specific ligands that interact with Newcastle disease virus (NDV). A fusion phage carrying the amino acid sequence TLTTKLY was selected from the panning procedure. An antibody competition assay showed that the selected phage was capable of competing with the polyclonal antibodies raised against NDV for binding sites on the virus. Determination of the binding affinity of this phage with NDV by an equilibrium binding assay in solution revealed two different dissociation constants, suggesting that there could be two distinct binding sites for the phage on NDV. Synthetic peptides with the sequence CTLTTKLYC, either in linear or cyclic conformations inhibited the binding of phage bearing the same sequence to NDV. These peptides also inhibited the hemolytic activity of the virus as well as its propagation in embryonated chicken eggs.