The Australian, heat-resistant, a virulent V4 strain of Newcastle disease (ND) virus was selected for further heat resistance to give a variant designated V4-UPM. V4-UPM was sprayed on to food pellets which were fed to chickens in amounts calculated to give about 10(6) EID50 per chicken. Chickens vaccinated only once by feeding developed no haemagglutination-inhibition (HI) antibodies and were not protected against challenge with a viscerotropic velogenic strain of ND virus. Chickens given food pellet vaccine at 3 and 6 weeks of age developed HI antibodies and were substantially protected against parenteral and contact challenge with virulent ND virus. Similar protection was achieved when the V4-UPM vaccine was given intranasally on two occasions or when the vaccine virus was allowed to spread by contact from intranasally vaccinated chickens to nonvaccinated chickens. Heat resistant ND vaccine incorporated in food pellets may provide a method for protecting village chickens against ND in tropical countries.
Meat chickens housed on a commercial broiler farm in Australia were vaccinated once at 10 to 11 days-of-age by aerosol with live V4 Newcastle disease virus (NDV) vaccine. Groups of vaccinated and unvaccinated birds were flown to Malaysia, where they were challenged with a virulent strain of NDV. Survival rates in vaccinated chickens challenged 7, 14, 21 or 31 d after vaccination were 0.47, 0.77, 0.97 and 0.92, respectively. All unvaccinated chickens died due to Newcastle disease (ND) following challenge. Chickens in Australia and Malaysia were bled and the serums tested for haemagglutination-inhibiting (HI) antibody to NDV. Many vaccinated birds with no detectable antibody, and all birds with a log2 titre of 2 or greater, survived challenge. The results showed that this V4 vaccine induced protective immunity in a significant proportion of chickens within 7 d of mass aerosol vaccination. This early immunity occurred in the absence of detectable circulating HI antibody. Non-HI antibody mediated immunity continued to provide protection up to 31 d after vaccination. Almost all vaccinated birds were protected within 3 w of vaccination. It is concluded that the V4 vaccine is efficacious and could be useful during an outbreak of virulent ND in Australia.
An erythromycin resistance plasmid, pAJ01 was isolated from Loctococcus lactis isolate C5 that was isolated from a healthy two-week-old chicken cecum. A 4 kb plasmid was transformed into plasmidless L. lactis MG1363 before a restriction endonuclease map was constructed. It was then fused with pUC19 to form pAJ02, which can replicate in Escherichia coli XLI-Blue as well as L. lactis MG1363. The plasmid was stably maintained in Lactococcus for more than 100 generations.
Newcastle disease virus (NDV) is one of the most economically important avian virus which affects the poultry industry worldwide. Although NDV is being very actively studied in Malaysia, there are still no studies on its potential as an anticancer agent, a new approach to treating cancer known as virotherapy. Currently, a collaborative research is being undertaken between Universiti Putra Malaysia (UPM), Universiti Sains Malaysia (USM) and Majlis Kanser Nasional (MAKNA) in characterising various local NDV isolates as anticancer agent. This paper describes an overview of the research that have been carried out worldwide in the use of NDV for cancer treatment and also some of our findings in characterising local NDVs with oncolytic properties.
Neoplastic transformation appears to be a multi-step process in which the normal controls of cell proliferation and cell-cell interaction are lost, thus transforming normal cells into cancer. The tumorigenic process involves the interplay between oncogenes and tumour suppressor genes. In this study, we have selected the ras family, c-myc and epidermal growth factor receptor (EGFR) genes to detect whether their abnormalities are associated with the expression and progression of glioma cases in Malay patients. We have used the polymerase chain reaction-single stranded conformation polymorphism followed by direct sequencing for the study. For the ras gene family, we screened the point mutations in codons 12 and 61 of the H-, K-, and N-ras gene; for EGFR and c-myc, we analyzed only the exon 1 in glioma samples. In mutational screening analyses of the ras family, c-myc and EGFR gene, there was no mobility shift observed in any tumour analyzed. All patterns of single stranded conformation polymorphism (SSCP) band observed in tumour samples were normal compared to those in normal samples. The DNA sequencing results in all high-grade tumours showed that all base sequences were normal. All 48 patients survived after five years of treatment. In simple logistic regression analysis, variables which were found to be significant were hemiplegia (p=0.047) and response radiotherapy (p=0.003). Hemiplegics were 25 times more likely to have high pathological grade compared to those without. Patients with vascular involvement were 5.5 times more likely to have higher pathological grade. However, these findings were not significant in multivariate analysis. Patients who had radiotherapy were nearly 14 times more likely to have higher pathological grade. Multivariate analysis revealed that patients with hemiplegia were more likely to have higher pathological grade (p= 0.008). Those with higher pathological grading were 80 times more likely to have radiotherapy (p=0.004).
The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specificpathogen-free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.
Brain tumorigenesis is a complex process involving multiple genetic alterations. Cyclin D1 and BAX genes are two of the most important regulators in controlling the normal proliferation and apoptosis of cells, respectively. In this study, we analysed the possibilities of involvement of cyclin D1 and BAX genes in the gliomagenesis.
House crows (Corvus splendens) in Selangor, Malaysia were examined for the presence of Campylobacter species, Salmonella species, Mycoplasma gallisepticum and Mycoplasma synoviae by serology, culture and pcr. For the detection of Campylobacter and Salmonella species swabs were taken either from the intestine or cloaca. For the detection of mycoplasmas, swabs were taken either from the choanal cleft or trachea for culture and pcr and serum samples were tested by the rapid serum agglutination (rsa) and monoclonal antibody-blocking elisa (mbelisa) for antibodies to M gallisepticum and M synoviae. For campylobacter, 25.3 per cent of the crows were positive by culture, and the species identified were Campylobacter jejuni and Campylobacter coli. No Salmonella species were isolated. Four of 24 swabs were positive for M gallisepticum dna but none gave positive results for M synoviae dna. No M gallisepticum or M synoviae antibodies were detected by rsa but 60 per cent of the sera gave positive reactions for M gallisepticum and 13 per cent gave positive reactions for M synoviae by mbelisa.
Avian influenza viruses are pathogens of economical and public health concerns. However, infections caused by low pathogenic avian influenza particularly H9N2 subtype are not associated with clear clinical features. Hence, rapid detection and subtyping of the virus will enable immediate measures to be implemented for preventing widespread transmission. This study highlights the development of a multiplex real-time reverse-transcriptase polymerase chain reaction (RRT-PCR) assay using SYBR Green 1 chemistry for universal detection of avian influenza viruses and specific subtyping of H9N2 isolates based on melting temperatures (T(m)) discriminations. Three melting peaks generated simultaneously at temperatures 85.2+/-1.0, 81.9+/-0.9 and 78.7+/-0.9 degrees C represent NP, H9 and N2 gene products, respectively. The RRT-PCR assay was about 10-100-fold more sensitive when compared to the conventional RT-PCR method using reference H9N2 isolate. In addition, the RRT-PCR assay was 100% sensitive as well as 92% specific according to the standard virus isolation method in detecting experimentally infected specific-pathogen-free (SPF) chickens.
Avian influenza viruses (AIV), the causative agent of avian flu or bird flu, cause widespread morbidity and mortality in poultry. The symptoms of the disease range from mild flu like symptoms to death. These viruses possess two important surface glycoproteins, namely hemagglutinin (HA) and neuraminidase (NA) against which neutralizing antibodies are produced. Due to the highly mutative nature of the genes which encode these proteins, the viruses often confer resistance to the current anti-viral drugs making the prevention and treatment of infection challenging. In our laboratory, we have recently identified a novel anti-viral peptide (P1) against the AIV H9N2 from a phage displayed peptide library. This peptide inhibits the replication of the virus in ovo and in vitro by its binding to the HA glycoprotein. In the current study, we demonstrate that the peptide inhibits the virus replication by preventing the attachment to the host cell but it does not have any effect on the viral fusion. The reduction in the viral nucleoprotein (NP) expression inside the host cell has also been observed during the peptide (P1) treatment. This novel peptide may have the potential to be developed as a therapeutic agent for the treatment and control of avian influenza virus H9N2 infections.
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.
A SYBR Green I based one-step real-time reverse transcriptase polymerase chain reaction was developed for the detection and differentiation of very virulent (vv) and classical strains of infectious bursal disease virus (IBDV). The assay showed high PCR efficiency >93% and high reproducibility with coefficient of variation less than 0.5%. When tested on characterized IBDV strains, the very virulent and classical-specific primers detected accurately only vvIBDV and classical IBDV strains, respectively. The diagnostic efficacy of the assay was also tested on 140 bursal samples from experimental infection and 37 bursal samples from cases suspected of IBD. The assay was able to detect IBDV from bursal samples collected at days 3 and 5 post-infection with the vvIBDV strain UPM94/273 and the classical IBDV strain D78. The assay was also able to detect bursal samples infected dually with D78 and UPM94/273. The melting temperature values of the amplification products from the classical and very virulent viral infection were statistically significant (P<0.05). The specificity of the assay for detecting IBDV from suspected cases was confirmed by sequence analysis of the VP2 gene. The assay showed high sensitivity since bursal samples which were negative for IBDV were confirmed by virus isolation and PCR amplification. Hence, the new assay offers an attractive method for rapid detection of strains of IBDV.
Avian influenza viruses (AIV) cause high morbidity and mortality among the poultry worldwide. Their highly mutative nature often results in the emergence of drug resistant strains, which have the potential of causing a pandemic. The virus has two immunologically important glycoproteins, hemagglutinin (HA), neuraminidase (NA), and one ion channel protein M2 which are the most important targets for drug discovery, on its surface. In order to identify a peptide-based virus inhibitor against any of these surface proteins, a disulfide constrained heptapeptide phage display library was biopanned against purified AIV sub-type H9N2 virus particles.
Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a highly contagious disease of birds and has been one of the major causes of economic losses in the poultry industry. Despite routine vaccination programs, sporadic cases have occasionally occurred in the country and remain a constant threat to commercial poultry. Hence, the present study was aimed to characterize NDV isolates obtained from clinical cases in various locations of Malaysia between 2004 and 2007 based on sequence and phylogenetic analysis of partial F gene and C-terminus extension length of HN gene.
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.
The aim of this study is to prepare a model for the production of Newcastle disease virus (NDV) lentogenic F strain using cell culture in bioreactor for live attenuated vaccine preparation. In this study, firstly we investigated the growth of Vero cells in several culture media. The maximum cell number was yielded by culture of Vero cells in Dulbecco's Modified Eagle Medium (DMEM) which was 1.93 x 10(6) cells/ml. Secondly Vero cells were grown in two-litre stirred tank bioreactor by using several commercial microcarriers. We achieved the maximum cell concentration about 7.95 x 10(5) cells/ml when using Cytodex 1. Later we produced Newcastle Disease virus in stirred tank bioreactor based on the design developed using Taguchi L4 method. Results reveal that higher multiplicity of infection (MOI) and size of cell inoculums can yield higher virus titer. Finally, virus samples were purified using high-speed centrifugation based on 3( * *)(3-1) Fractional Factorial Design. Statistical analysis showed that the maximum virus titer can be achieved at virus sample concentration of 58.45% (v/v), centrifugation speed of 13729 rpm, and centrifugation time of 4 hours. As a conclusion, high yield of virus titer could be achieved through optimization of cell culture in bioreactor and separation by high-speed centrifugation.
To explore the effects of the combination of tryptophan (Trp) and arginine (Arg) on growth performance, serum parameters and immune response of broiler chickens challenged with intermediate plus strain of infectious bursal disease virus vaccine, an in vivo experiment was conducted. A corn-soybean meal-based diet containing different levels of Arg and Trp was used. Cobb500 male broiler chickens from 0 to 49 days of age were subjected to a diet supplemented with the combination of Trp and Arg. Growth performance parameters and serum parameters were measured at 27 and 49 days of age. To evaluate the immunomodulatory effects of the combination of Trp and Arg on the challenged chickens, we measured the serum levels of interferon-α, interferon-γ and immunoglobulin G at 27, 35, 42, and 49 days of age. The results showed that the three evaluated immune system parameters including interferon-α, interferon-γ and immunoglobulin G were significantly enhanced after treatment. This enhancement resulted in the recovery of infectious bursal disease virus-infected chickens compared with controls as confirmed by histopathological examinations. Moreover, serum parameters such as albumin and total protein increased, whereas the treatment decreased (P<0.05) the feed:gain ratio, aspartate amino-transferase, alkaline phosphatase, lactic dehydrogenase, triglyceride and cholesterol. These findings suggest that the combination of Arg and Trp has a regulatory effect on growth performance. Moreover, it modulates the systemic immune response against infectious bursal disease.
Newcastle disease virus (NDV) is a member of genus Avulavirus within the family Paramyxoviridae. Interest of using NDV as an anticancer agent has arisen from its ability to kill tumor cells with limited toxicity to normal cells. In this investigation, the cytotolytic properties of NDV strain AF2240 were evaluated on brain tumor cell line, anaplastic astrocytoma (U-87MG), by using MTT assay. Cytological observations were studied using fluorescence microscopy and transmission electron microscopy to show the apoptogenic features of NDV on U-87MG. DNA laddering in agarose gel electrophoresis and terminal deoxyribonucleotide transferase-mediated dUTP-X nick end-labeling staining assay confirmed that the mode of cell death was by apoptosis. However, analysis of the cellular DNA content by flowcytometery showed that there was a loss of treated U-87MG cells in all cell cycle phases (G1, S and G2/M) accompanied with increasing in sub-G1 region (apoptosis peak). Early apoptosis was observed 6 h post-inoculation by annexin-V flow-cytometry method. It could be concluded that NDV strain AF2240 is a potent antitumor agent that induce apoptosis and its cytotoxicity increasing while increasing of time and virus titer.
Newcastle disease virus (NDV) is used as an antineoplastic agent in clinical tumor therapy. It has prompted much interest as an anticancer agent because it can replicate up to 10,000 times better in human cancer cells than in most normal cells. This study was carried out to determine the oncolytic potential of NDV strain AF2240 and V4-UPM on WEHI-3B leukemia cell line. Results from MTT cytotoxicity assay showed that the CD(50) values for both strains were 2 and 8 HAU for AF2240 and V4-UPM, respectively. In addition, bromodeoxyuridine (BrdU) and trypan blue dye exclusion assays showed inhibition in cell proliferation after different periods. Increase in the cellular level of caspase-3 and detection of DNA laddering using agarose gel electrophoresis on treated cells with NDV confirmed that the mode of cell death was apoptosis. In addition, flow-cytometry analysis of cellular DNA content showed that the virus caused an increase in the sub-G1 region (apoptosis peaks). In conclusion, NDV strains AF2240 and V4-UPM caused cytolytic effects against WEHI-3B leukemic cell line.
Newcastle disease virus (NDV) is a member of the Paramyxoviridae that has caused severe economic losses in poultry industry worldwide. Several strains of NDV were reported to induce cytolysis to cancerous cell lines. It has prompted much interest as anticancer agent because it can replicate up to 10,000 times better in human cancer cells than in most normal cells. In this study, two NDV strains, viserotropic-velogenic strain AF2240 and lentogenic strain V4-UPM, showed cytolytic activity and apoptosis induction against Mouse myelomoncytic leukemia (WEHI 3B). The cytolytic effects of NDV strains were determined using microtetrazolium (MTT) assay. The cytolytic dose - fifty percent (CD(50)) were 2 and 8HAU for AF2240 and V4-UPM strains, respectively. Cells treated with NDV strains showed apoptotic features compared to the untreated cells under fluorescence microscope. NDV induced activation of caspase-3 and DNA laddering in agarose gel electrophoresis which confirmed the apoptosis. The anti-leukemic activity of both strains was evaluated on myelomoncytic leukemia BALB/c mice. The results indicated that both NDV strains significantly decreased liver and spleen weights. It also decreased blasts cell percentage in blood, bone marrow and spleen smears of treated mice (p<0.05). Histopathological studies for spleen and liver confirmed the hematological results of blood and bone marrow. From the results obtained, the exposure to both NDV stains AF2240 and V4-UPM showed similar results for Ara-c. In conclusion NDV strains AF2240 and V4-UPM can affect WEHI 3B leukemia cells in vitro and in vivo.