Newcastle Disease Virus LaSota Strain Research Articles

Newcastle disease virus LaSota strain induces apoptosis and activates the TNFα/NF-κB pathway in canine mammary carcinoma cells.

Spontaneous canine mammary carcinomas (CMCs) have been widely considered a good research model for human breast cancers, which brings much attention to CMCs. In recent years, the oncolytic effect of Newcastle disease virus (NDV) on cancer cells has been widely studied, but its effect on CMCs is still unclear. This study aims to investigate the oncolytic effect of NDV LaSota strain on canine mammary carcinoma cell line (CMT-U27) in vivo and in vitro. The in vitro cytotoxicity and immunocytochemistry experiments showed that NDV selectively replicated in CMT-U27 cells, and inhibited cell proliferation and migration but not in MDCK cells. KEGG analysis of transcriptome sequencing indicated the importance of the TNFα and NF-κB signalling pathways in the anti-tumour effect of NDV. Subsequently, the significantly increased expression of TNFα, p65, phospho-p65, caspase-8, caspase-3 and cleaved-PARP proteins in the NDV group suggested that NDV induced CMT-U27 cells apoptosis by activating the caspase-8/caspase-3 pathway and the TNFα/NF-κB signalling pathway. Nude mice tumour-bearing experiments showed that NDV could significantly decrease the growth rate of CMC in vivo. In conclusion, our study demonstrates the effective oncolytic effects of NDV on CMT-U27 cells in vivo and in vitro, and suggests NDV as a promising candidate for oncolytic therapy.

Efficient Production of Evolutionary Nanoparticle Newcastle Disease Virus

Viruses are routinely isolated from infected cells through freeze–thaw (F–T) cycles or sonication. The aim of this study was to compare different methods for efficient isolation of Newcastle disease virus (NDV) particles from BSR-T7/5 (BSR) cells. The BSR cells were infected with NDV LaSota strain, and the virus particles were isolated via F–T, sonication, sonication followed by F–T, and F–T followed by sonication. The infection and proliferation kinetics of the virus were analyzed by cytopathic observation and monitoring of hemagglutination (HA) titers. The virus isolated by sequential F–T and sonication was amplified through five passages of BSR cells, and then used to infect the HepG2 cells. The viability and apoptosis rates of the infected cells were evaluated by Cell Counting Kit-8 assay and Annexin V-FITC/PI staining respectively. We successfully obtained NDV particles from persistently infected BSR cells through all four methods, which indicated that the LaSota can effectively replicate in BSR cells. However, F–T followed by sonication was optimum in terms of separation effect. The virus particles isolated by this method still exhibited cytolytic activity against HepG2 cells. Thus, our novel method can be applied to NDV production and generation of tumor vaccines.

Evaluation of Newcastle disease virus LaSota strain attenuated by codon pair deoptimization of the HN and F genes for in ovo vaccination

In ovo vaccination is an attractive immunization approach for the poultry industry. However, commonly used Newcastle disease virus (NDV) vaccines cannot be administered in ovo because of the reduced hatchability and embryo mortality. The codon pair deoptimization (CPD) approach has been used to efficiently and rapidly attenuate viruses by targeting the virulence genes. In this study, we aimed to attenuate the NDV LaSota (LS) vaccine strain for in ovo vaccination by CPD of the fusion (F) or/and hemagglutinin-neuraminidase (HN) genes with approximately 44 % suboptimal codon substitutions. Three NDV LS recombinants expressing codon deoptimized F (rLS/F-d), HN (rLS/HN-d), or both genes (rLS/F+HN-d) were generated using reverse genetics technology. Biological assays showed that the CPD viruses retained similar hemagglutination activity and growth ability to the parental rLS virus. The CPD of the HN gene slightly attenuated the rLS/HN-d and rLS/F+HN-d viruses, whereas the CPD of the F gene marginally increased the rLS/F-d virus pathogenicity compared to rLS. Nevertheless, all three CPD rLS viruses were still lethal to 10-day-old specific-pathogen-free (SPF) chicken embryos. In ovo inoculation of 18-day-old SPF chicken embryos with the CPD viruses severely reduced chicken’s hatch and survival rates. These results suggested that the CPD of the surface glycoprotein genes of the LS strain at the current level of suboptimal codon substitutions could not sufficiently attenuate the virus for use as an in ovo vaccine, and codon deoptimizing a greater proportion of the F and HN genes or additional gene(s) may be required for sufficient attenuation of the LS strain.

The Potential Selective Oncolytic Effects of Newcastle Disease Virus (NDV) on Cervical and Colon Cancer Cell Lines

The mesogenic and velogenic strains of the avian Newcastle Disease Viruses (NDV) have been investigated for use in cancer virotherapy. However, despite its promising results, these strains have the potential to cause outbreaks leading to morbidity and mortality in large numbers of avians that can devastate the poultry industry. Therefore, there is a growing interest towards the use of lentogenic strains such as NDV LaSota vaccine strain for virotherapy. This study investigated the oncolytic potential of the NDV LaSota strain on colorectal cancer (HT29) and cervical cancer (HeLa) cell lines. The NDV LaSota stain was propagated in pathogen free embryonated chicken eggs and quantified using a hemagglutination assay. The oncolytic activity was evaluated by comparison of HT29 and HeLa cell lines to the non-cancerous human embryonic kidney (HEK293) cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay. The highest NDV concentration of 16 HAU was found to reduce the viability of HT29, HeLa, and HEK293 cell lines to 42±3% (p = 0.0001), 49±12% (p = 0.0009), and 77±5% (p = 0.007), respectively, indicating its potential selective cytotoxicity, as supported by the disturbed cell morphology. The NDV LaSota strain exhibits a potential selective oncolytic activity towards cancer cell lines, thus may serve as an interesting candidate for virotherapy against colorectal and cervical cancer.

Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as a live virus vaccine candidate.

BackgroundDue to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus.MethodsHere, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type format or a membrane-anchored format lacking the polybasic cleavage site. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, the immunogenicity and protective efficacy of these NDV-based vaccines were investigated.FindingsWe report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly in mice. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs.InterpretationThe results suggested that the NDV vector expressing either the wild type S or membrane-anchored S without the polybasic cleavage site could be used as live vector vaccine against SARS-CoV-2.FundingThis work is supported by an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS) contract, the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract, philanthropic donations and NIH grants.

Evaluation of Apoptosis Induction by Newcastle Disease Virus LaSota Strain in Human Breast Carcinoma Cells.

The innovation of therapeutic modalities with better clinical efficacy is necessary for the treatment of patients with advanced cancers. Newcastle disease virus (NDV), an avian pathogenic virus, is one of the most promising oncolytic viruses that can replicate selectively in human cancer cells. In humans, NDV can cause transient conjunctivitis and mild flu-like symptoms. However, this virus poses no hazard to human health. The elucidation of the mechanisms of cancer cell killing by NDV is helpful for the clinical application of NDV in cancer patients. Regarding this, the present study was performed to evaluate apoptosis induction by NDV LaSota strain vaccine in human breast carcinoma cells. To this end, MCF-7 cells, a human breast adenocarcinoma cell line, were infected with NDV in vitro. Tumor cell cytotoxicity, apoptosis induction, and expression levels of apoptosis-related genes were examined in NDV-infected breast carcinoma cells. Tumor cell cytotoxicity was measured by 3-[4,5-dimethylthiazol-2yl]2,5-diphenyl-tetrazoliumbromide (MTT) assay. The induction of apoptosis was assessed by annexin V/propidium iodide staining. In addition, the expression levels of apoptosis-related genes were evaluated using real-time reverse transcription polymerase chain reaction (PCR) technique. The NDV showed cytotoxic effects on MCF-7 cells and induced apoptosis in the infected carcinoma cells. The gene expression levels of BAX, caspase-9, and caspase-3, but not BAK-1, were increased in NDV-infected cancer cells, compared to the gene expression levels in the non-infected cancer cells. These results suggest that the induction of the intrinsic pathway of apoptosis is one of the mechanisms that can contribute to cancer cell killing by NDV. Additional studies are required to investigate other probable mechanisms involved in NDV-mediated cancer cell killing.

In Vitro Oncolytic activity of non-virulent Newcastle Disease Virus LaSota Strain against Mouse mammary adenocarcinoma

Newcastle disease virus (NDV) is a wide-spectrum anti-tumor agent. The oncolytic selectivity of NDV, a family of Paramyxoviridae, depends on the differential type of inducing different death pathways. This work was conducted to further understand the oncolytic effect of LaSota strain. A mouse breast cancer model (Murine mammary adenocarcinoma cell line AMN3) was used in this study. Methyl Thiazolyl Tetrazolium (MTT) viability assay tested different NDV multiplicity of infection (MOI) values on mouse mammary adenocarcinoma cells incubated for 72 hours post-infection. The IC50 values and anti-tumor activity of LaSota strain against AMN3 cell line were determined. Following Hematoxylin and Eosin Stain, we examined the morphological modifications of IC50 along with 10 MOI values of NDV. The induction of NDV apoptosis in AMN3 cells was investigated using the technique of staining acridine orange and propidium iodide (AO / PI). Immunocytochemistry assay was performed using anti-NDV mAbs and caspases 8 and 9 to study NDV replication and apoptosis induction mechanisms. The lentogenic LaSota NDV strain, a live vaccine, demonstrated the oncolytic effect on mammary cancer cells of the AMN3 mouse and showed that LaSota strain triggered a dose-dependent increase in infected cells’ apoptosis relative to untreated mammary cancer cells. The immunocytochemistry study showed that NDV infected cells were positive for virus infection and that caspase9 in mouse mammary cancer cells after LaSota strain infection was significantly enhanced compared to caspase 8. In conclusions, NDV LaSota strain had oncolytic effects by destroying tumor cells and triggering the intrinsic apoptosis pathways in mouse mammary cancer cells. However, the mechanisms of the in vivo anti-tumor activity need to be better understood.

Studies on immune response to Newcastle disease virus in broiler chickens fed with Lactobacillus reuteri PIA16 isolated from the gut of indigenous chicken of Assam, India.

Background and Aim:The chicken gut harbors microflora which impacts the health, production performance and immune response against pathogens. Assam local chickens reared under natural conditions are known to possess high immunocompetence which may be attributable to its gut microbiota make-up. This study aimed to investigate the individual effect of two strains of Lactobacillus reuteri PIA16 isolated separately from cecum and jejunum of Assam indigenous chicken on the immunity of broiler chickens against Newcastle disease virus (NDV) when fed singly and in combination with a prebiotic.Materials and Methods:A total of 240 birds (48 per group) were vaccinated with Lasota strain of NDV on the 5th and 21st day of age. Blood samples were collected before and after immunization against ND for the detection of humoral antibody response by hemagglutination inhibition test. The cell-mediated immune (CMI) response was estimated through response to phytohemagglutinin-P (PHA-P) and expressed as web index.Results:A significant influence on the immune response to NDV was observed in all the L. reuteri PIA16 as well as mannan oligosaccharide (MOS) supplemented groups revealing higher antibody titer than the control counterpart. The CMI response revealed a better cutaneous basophilic hypersensitivity response to PHA-P in the treated groups than the control.Conclusion:Enhancement in immunity was perceived in the broilers fed with L. reuteri PIA16 and in combination with MOS due to the stimulation of the host’s humoral and CMI response by the probiotics and prebiotics used.

Evaluation of the effect of live LaSota Newcastle disease virus vaccine as primary immunization on immune development in broilers

Newcastle disease remains a major concern to the poultry industry; however, it can be managed with effective vaccination programs. The main objective of this study was to evaluate the effect of 2 1× doses of live LaSota strain Newcastle disease virus (NDV) vaccine administered oculo-nasally on d one and 21 on development of humoral and cell-mediated immune response in broilers, and to compare different immunization schedules. Two experiments were conducted. In Experiment I (n = 320), Ross 308 birds were randomly assigned to an unvaccinated control group or vaccinated treatment. [Both treatments consisted of 4 pens per treatment and 40 birds per pen]. At d one, live NDV LaSota strain vaccine was used as a primary immunization to evaluate its impact on adaptive immunity. No substantial NDV-specific humoral immune response was established. Body weights were significantly higher (P < 0.05) in the vaccinated birds on d 4 and 7. Spleen index of the vaccinated birds was significantly (P < 0.05) lower at d 28 and 35. Flow cytometry showed reduced levels of peripheral and splenic B and T lymphocytes. Interferon gamma secreted by splenocytes and in circulation was measured; the results showed a reduced expression post-secondary immunization. In Experiment II (n = 180), the role of maternal antibodies and primary vaccination at d one was evaluated using 3 vaccination protocols. Protocol 1 used live B1 strain as primary immunization, whereas protocol 2 used live LaSota strain. Protocol 3 used live LaSota strain after maternal antibodies had decayed. Protocol 3 resulted in the highest NDV-titer level during the trial. Protocol 2 had the lowest NDV titer. Feed conversion was significantly higher (P < 0.05) in protocol 3 compared to 1 and 2. Overall, the results indicate that the use of live LaSota strain NDV vaccine as primary immunization at d one has a detrimental effect on the development of adaptive immunity in broilers; however, its use after the level of maternal antibodies decays results in a robust antigen-specific humoral immune response.

In Vitro Synergistic Enhancement of Newcastle Disease Virus to 5-Fluorouracil Cytotoxicity against Tumor Cells.

Background: Chemotherapy is one of the antitumor therapies used worldwide in spite of its serious side effects and unsatisfactory results. Many attempts have been made to increase its activity and reduce its toxicity. 5-Fluorouracil (5-FU) is still a widely-used chemotherapeutic agent, especially in combination with other chemotherapies. Combination therapy seems to be the best option for targeting tumor cells by different mechanisms. Virotherapy is a promising agent for fighting cancer because of its safety and selectivity. Newcastle disease virus is safe, and it selectively targets tumor cells. We previously demonstrated that Newcastle disease virus (NDV) could be used to augment other chemotherapeutic agents and reduce their toxicity by halving the administered dose and replacing the eliminated chemotherapeutic agents with the Newcastle disease virus; the same antitumor activity was maintained. Methods: In the current work, we tested this hypothesis on different tumor cell lines. We used the non-virulent LaSota strain of NDV in combination with 5-FU, and we measured the cytotoxicity effect. We evaluated this combination using Chou–Talalay analysis. Results: NDV was synergistic with 5-FU at low doses when used as a combination therapy on different cancer cells, and there were very mild effects on non-cancer cells. Conclusion: The combination of a virulent, non-pathogenic NDV–LaSota strain with a standard chemotherapeutic agent, 5-FU, has a synergistic effect on different tumor cells in vitro, suggesting this combination could be an important new adjuvant therapy for treating cancer.

Generation of Newcastle Disease Virus (NDV) Recombinants Expressing the Infectious Laryngotracheitis Virus (ILTV) Glycoprotein gB or gD as Dual Vaccines.

Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infection with infectious laryngotracheitis virus (ILTV), a member of the family Herpesviridae. The current commercial ILT vaccines are either unsafe or ineffective. Therefore, there is a pressing need to develop safer and more efficacious vaccines. Newcastle disease (ND), caused by infection with Newcastle disease virus (NDV), a member of the family Paramyxoviridae, is one of the most serious infectious diseases of poultry. The NDV LaSota strain, a naturally occurring low-virulence NDV strain, has been routinely used as a live vaccine throughout the world. This chapter describes the generation of Newcastle disease virus (NDV) LaSota vaccine strain-based recombinant viruses expressing glycoprotein B (gB) or glycoprotein D (gD) of ILTV as dual vaccines against ND and ILT using reverse genetics technology.

Apoptotic induction of lung adenocarcinoma A549 cells infected by recombinant RVG Newcastle disease virus (rL-RVG) in vitro.

Newcastle disease virus (NDV) is a member of the genus Avulavirus in the Paramyxoviridae family and its antitumor properties depend on its ability to kill malignant cells while not affecting normal cells. The present study investigated a recombinant avirulent NDV LaSota strain (wild-type NDV strain) expressing the rabies virus glycoprotein (rL-RVG), examined its oncolytic effect on the lung adenocarcinoma A549 cell line and evaluated its potential to serve as a vaccine against lung cancer. A549 cells were infected with the rL-RVG virus and analyzed by MTT, western blot, polymerase chain reaction (PCR), immunofluorescence, terminal deoxynucleotidyl transferase dUTP nick end labeling and flow-cytometric analyses. PCR, western blot and immunofluorescence showed that the RVG gene and protein were stably expressed in A549 cells following infection with rL-RVG. The growth of A549 cells in the rL-RVG group was inhibited more effectively compared to those infected with the wild-type NDV strain. MTT results showed that cell growth inhibition rates in the rL-RVG group were significantly higher than those in the NDV group (P<0.05). Early apoptosis in the rL-RVG group was also more evident, with the apoptotic index being increased in rL-RVG group. The expression of the pro-apoptotic proteins caspase-3, -8 and -9 increased. The expression of caspase-3 decreased following application of the broad-specificity caspase inhibitor Z-VAD-FMK. However, the expression of the inhibitory apoptosis protein B-cell lymphoma 2 (bcl-2) did not change, but bcl-2-associated X/bcl-2 ratio was higher in the rL-RVG group than that in the NDV group. The rL-RVG strain was able to suppress lung cancer cell growth and promote lung cancer cell apoptosis to a greater extent than the wild-type NDV strain. Therefore, the rL-RVG strain is a potent antitumor agent.

Genetically engineered Newcastle disease virus expressing interleukin 2 is a potential drug candidate for cancer immunotherapy

Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, several clinical trials have reported that mesogenic NDV is a safe and effective agent for human cancer therapy. Interleukin 2 (IL2) is a cytokine that stimulates T cell propagation to trigger innate and adaptive immunity. IL2 has been used for cancer therapy and has achieved curative effects. In this study, a recombinant NDV LaSota strain expressing human interleukin 2 (rLaSota/IL2) was generated. The ability of rLaSota/IL2 to express human IL2 was detected in the infected tumor cells. In addition, the activity of IL2 was analyzed. The antitumor potential of rLaSota/IL2 was studied by xenograph mice carrying H22 and B16-F10 cells. Tumor-specific CD4+ and CD8+ T cells and MHC II were also analyzed in the two tumor-bearing models. Our study showed that rLaSota/IL2 significantly stimulated tumor-specific cytotoxic T-lymphocyte (CTL) responses and increased regulatory CD4+ and cytotoxic CD8+ T cells proliferation. The treatment with rLaSota/IL2 led to tumor regression in tumor-bearing mice and prolonged the survival of tumor-bearing mice. Furthermore, tumor challenging experiments demonstrated that rLaSota/IL2 invoked mice a unique capacity to remember a pathogen through the generation of memory T cells, which protect the host in the event of reinfection and form adaptive immune system. The result indicates that tumor-infiltrating CD4+ T regulatory cells may denote the effective regression of tumors. Taken together, rLaSota/IL2 has potential for immunotherapy and oncolytic therapy of cancers and may be an ideal candidate for clinical application in future cancer therapy.

Ascaridia galli infection influences the development of both humoral and cell-mediated immunity after Newcastle Disease vaccination in chickens

Potent vaccine efficiency is crucial for disease control in both human and livestock vaccination programmes. Free range chickens and chickens with access to outdoor areas have a high risk of infection with parasites including Ascaridia galli, a gastrointestinal nematode with a potential influence on the immunological response to vaccination against other infectious diseases. The purpose of this study was to investigate whether A. galli infection influences vaccine-induced immunity to Newcastle Disease (ND) in chickens from an MHC-characterized inbred line. Chickens were experimentally infected with A. galli at 4 weeks of age or left as non-parasitized controls. At 10 and 13 weeks of age half of the chickens were ND-vaccinated and at 16 weeks of age, all chickens were challenged with a lentogenic strain of Newcastle disease virus (NDV). A. galli infection influenced both humoral and cell-mediated immune responses after ND vaccination. Thus, significantly lower NDV serum titres were found in the A. galli-infected group as compared to the non-parasitized group early after vaccination. In addition, the A. galli-infected chickens showed significantly lower frequencies of NDV-specific T cells in peripheral blood three weeks after the first ND vaccination as compared to non-parasitized chickens. Finally, A. galli significantly increased local mRNA expression of IL-4 and IL-13 and significantly decreased TGF-ß4 expression in the jejunum two weeks after infection with A. galli. At the time of vaccination (six and nine weeks after A. galli infection) the local expression in the jejunum of both IFN-? and IL-10 was significantly decreased in A. galli-infected chickens. Upon challenge with the NDV LaSota strain, viral genomes persisted in the oral cavity for a slightly longer period of time in A. galli-infected vaccinees as compared to non-parasitized vaccinees. However, more work is needed in order to determine if vaccine-induced protective immunity is impaired in A. galli-infected chickens.

Cell-Type-Specific Innate Immune Response to Oncolytic Newcastle Disease Virus

Virotherapy of cancer exploits the potential of naturally occurring and engineered oncolytic viruses to selectively replicate in and cause cytotoxicity to tumor cells without affecting healthy normal cells. The tumor selectivity of Newcastle disease virus (NDV), a member of the family Paramyxoviridae, depends on the differential type I interferon (IFN) response. Further understanding of the key mechanisms and immune effector molecules involved will aid in augmenting the oncolytic properties of NDV. Here we report on the infection kinetics and innate immune responses to a recombinant LaSota strain of NDV (rLaSota eGFP) in human tumor and normal cells. We observed varying replicative fit and cytotoxicity of rLaSota eGFP depending on the tumor cell type, with severely restricted replication in normal cells. The absence of retinoic acid-inducible gene I (RIG-I), a cytosolic RNA sensor, determined sensitivity to NDV. Productive NDV infection with a moderate IFN-α induction in human multiple myeloma cells suggested a role for IFN-independent mechanisms or lack of type I IFN reinforcement by RIG-I. Proinflammatory cytokines and chemokines were altered differentially in infected normal and tumor cells. Our results suggest that tumor selectivity is dependent on variations in the cellular antiviral response to infection with NDV and RIG-I expression.

EFECTIVIDAD DE LA VACUNA CONTRA LA ENFERMEDAD DE NEWCASTLE EN EL CONTROL DE LA PAPILOMATOSIS BOVINA

The efficacy of Newcastle disease virus La Sota strain (NDV-LS) attenuated vaccine for treating bovine papillomatosis was evaluated. A total of 64 crossbreed female dairy cattle with clinically diagnosed skin papillomatosis, reared in farms of Alto Mayo, San Martín, Peru were used in the study. A group of 34 animals were subcutaneously inoculated with 2 ml of NDV-LS attenuated vaccine (109 infective dose50 in egg) on day 0 and day 7, and were clinically evaluated on days 0, 7, 30, 45, and 60 after inoculation. The remainderanimals were kept as controls. Effective treatment was considered when at least 75% of papillomas successfully underwent to regression. Evaluation on day 60 showed the treatment to be effective in three animals (8.8%). No statistical difference was found with the control group. No secondary signs were observed in treated animals during the study. In conclusion, La Sota strain attenuated vaccine against Newcastle disease was not effective in treating and controlling bovine papillomatosis in semi-extensively managed animals from Alto Mayo region.

Validation of a Real-Time RT-PCR Method to Quantify Newcastle Disease Virus (NDV) Titer and Comparison with Other Quantifiable Methods

A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 (TCID50) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity.

Transient expression and identification of gene P and NP of NDV LaSota strain in two different cells

To Construction of P and NP genes eukaryotic expression vectors of Newcastle Disease Virus LaSota strain,study its reverse genetics and functional genome of NDV. P, NP genes were amplified and cloned into pGEM-T easy vector and then subcloned into pcDNA3.1 (+) expression vector respectively, the recombinant plasmids were named pcDNA3.1 (+)-P and pcDNA3.1 (+)-NP, Recombinant plasmids were transfected into 293 and BHK-21 cells respectively and were detected using IE and Western blot analysis. Expression of P, NP genes were detected and confirmed by the IE and WB analysis. The recombinant eukaryotic plasmids pcDNA3. 1(+)-P, pcDNA3.1 (+)-NP were expressed in 293 and BHK-21 cells successfully. This research may be helpful for further study of reverse genetics and functional genome of NDV.

The Effect on Pathogenesis of Newcastle Disease Virus LaSota Strain from a Mutation of the Fusion Cleavage Site to a Virulent Sequence

The principal molecular determinant of virulence of Newcastle disease virus (NDV) is the amino acid sequence at the fusion cleavage activation site. To extend the understanding of the role of the fusion cleavage activation site in NDV virulence, the pathogenesis in chickens of a lentogenic LaSota isolate and two infectious clones, NDFL and NDFLtag, were compared. NDFL is an infectious clone of a lentogenic NDV strain (LaSota E13-1), and NDFLtag is the infectious clone with the fusion cleavage site sequence mutated to the virulent motif. NDFL and NDFLtag were described by Peeters et al. The viruses were inoculated intraconjunctivally into groups of 4-wk-old white leghorn chickens and compared in a pathogenesis study for determination of disease causation (clinical signs of disease, gross lesions, histology, virus isolation, and serology) and viral distribution (presence of viral nucleoprotein and mRNA was detected by immunohistochemistry and in situ hybridization, respectively). The modification of the fusion cleavage activation site to the virulent motif in the infectious clone only slightly increased disease severity and viral distribution in the pathogenesis assessment, even though dramatically increased pathogenicity of NDFLtag was confirmed by standard pathogenicity index tests. The result, that the mutated fusion cleavage site of NDV-NDFLtag had only a small influence on pathogenesis in chickens compared to either E13-1 or NDFL, suggests that the pathogenic effects of NDV are not dependent on the fusion cleavage site alone.

FC‐16 Clinical and immunological effects of Newcastle disease virus vaccine on bovine papillomatosis

Newcastle disease virus (NDV) has antineoplastic and immunostimulatory properties, and it is currently being clinically tested in anticancer therapy. In order to analyse the immunostimulatory effects of NDV on bovine papillomatosis, we inoculated 14 cows subcutaneously with an attenuated vaccine containing the LaSota strain of NDV (LS‐NDV). Four cows with papillomatosis served as controls. Serum samples were collected from each animal 1 h preinoculation and 7 and 21 days postinoculation. In inoculated cows on days 7 and 21, the mean antibody titres were log2 2.43 ± 0.92 and 5.57 ± 0.72, respectively, by haemagglutination inhibition, and the mean levels of TNFα were 5.80 ± 4.19 and 5.39 ± 2.66 ng/mL, respectively, by WEHI‐164 cytotoxicity assay. Significant differences between inoculated and control animals were evident for antibody titres on day 21 and clinical scores on day 60. A correlation was evident between the TNFα activities and clinical scores on day 21. The clinical observations at day 60 showed that the papillomas in five cows had completely resolved (36%), one animal had no alterations on clinical appearance of the tumour (7%), and papillomas in eight cows had regressed (57%). In conclusion, these results demonstrated that inoculation of LS‐NDV vaccine stimulates an antibody response and a limited increase in TNFα activity and may enhance clinical recovery in bovine papillomatosis. Funding: Scientific Research Council of Akdeniz University.