Newcastle Disease Virus Vaccine Research Articles

Molecular Detection and Characterization of Newcastle Disease Virus from Chickens in Mid-Rift Valley and Central Part of Ethiopia.

Newcastle disease (ND) is a highly infectious poultry disease that causes major economic losses worldwide. The disease is caused by Newcastle Disease Virus (NDV) and early detection and identification of the viral strain is essential. Having knowledge of the NDV strain genotype that circulates in some regions would help in designing an effective vaccine to control the disease. In this regard, there is little information on NDV strain in chickens in mid Rift Valley and the central part of Ethiopia. Therefore, the purpose of this study was to detect and characterize NDV strain genotype from chickens in mid-Rift Valley and the central part of Ethiopia and test whether this NDV strain genotype matches the vaccine strain currently used in the study area. A total of 98 samples: 78 (tracheal and cloacal) swabs from chicken pools of five and 20 tissue samples were collected. To detect NDV strain, conserved region of the virus Matrix (M) gene was amplified by qRT-PCR. To characterize NDV strain genotypes, M-gene positive samples were specifically re-amplified by conventional PCR targeting the Fusion (F) gene region and sequenced by Sanger method. 13.26% of tested samples were positive for NDV strain in the study area with statistically significant difference (P<0.05) among the study sites. Further characterization of the F genes from NDV strain isolates by phylogenetic analysis indicated that one field isolate clustered with genotype VII whereas three of the isolates clustered to genotype I, II, and III. The isolate of the current NDV strain vaccine in use in the study area clustered with genotype II. The current study indicates the existence of different NDV strain genotype from that of the vaccine strain currently used. Even though large-scale characterization of several isolates is required at national level, the current study laid baseline information for the existence of variations between field NDV strain genotype and vaccine strain currently used against ND in the country.

Expression of haemagglutinin-neuraminidase of newcastle virus genotype VIII in insect cell Sf9

The hemagglutinin-neuraminidase (HN) protein is the surface glycoprotein of Newcastle disease virus (NDV) which is involved in the process of virus entry into host cells. HN exhibits the capability to induce agglutination of chicken red blood cells, thereby contributing to the viral infection of the host. As a consequence, HN is a potential antigen for the production of subunit vaccines for protecting poultry from NDV infections. In this study, the hn gene coding for the HN antigen of NDV genotype VIII was artificially synthesized, ligated into donor vector pFastBacHT A and integrated into the baculovirus genome for the purpose of HN expression in Spodoptera frugiperda 9 (Sf9) cells. The recombinant HN protein was successfully expressed in Sf9 cells in the SF900 III medium. The expressed recombinant HN protein, with a size of approximately 63 kDa, exhibited hemagglutinin activity (HA) of 4log2 when interacting with chicken erythrocyte fluid. The findings of this study lay the foundation for subsequent research endeavors aimed at the development of Newcastle disease virus (NDV) vaccines tailored for poultry in Vietnam.

Evaluation of the immune response of layer chickens to Newcastle disease virus vaccines using the new vaccination regimens.

The study aimed to evaluate the immunological response of layer chickens to live Newcastle disease virus vaccine using a newly developed vaccine schedule administered via the ocular route, as well as assess the persistence of passive antibodies in layer chickens and the effectiveness of protection against strains of the virus. A total of 140-day-old Lohmann Brown chicks were randomly divided into seven groups, 20 chicks each. Groups 1-3 received a single eye instillation of the vaccine at ages 5, 26 and 54 days, respectively, whereas groups 4-6 received a double eye instillation. Group 7 served as non-vaccinated control group. Ten days after immunization, samples were taken from hens that had received the vaccine at ages 15, 36 and 64, as well as from control chickens that had not received the vaccine at ages 5, 15, 21 and 31. A total of 10 serum samples from all chickens exhibited protective antibodies, and booster doses resulted in the highest haemagglutination inhibition titre. No significant change in antibody production was observed among layer hens (p>0.05). The study found that the La Sota (GMT±SD: 6.71±4.96), La Sota (GMT±SD: 8.00±0.00) and thermostable I2 (GMT±SD: 7.60±6.02), vaccination schedules provided the maximum immune response in single eye instillation, whereas the HB1 (GMT±SD: 7.11±4.77), La Sota (GMT±SD: 7.83±5.76) and La Sota (GMT±SD: 7.60±6.02), combination was the second-best vaccination schedule in double eye instillation. Furthermore, maternally-derived antibodies were maintained up to 31 days of age, indicating the level of passive immunity prior to vaccination. Characteristic lesions, such as edematous and diphtheria mucosal membranes of the trachea, along with petechial and necrotic haemorrhages of the proventriculus, were observed during the necropsy of the birds that died from the challenged virus. This study suggests that subsequent live virus vaccine by ocular route immunization is required to effectively protect against velogenic viscerotropic Newcastle disease infection. The results also highlight the importance of developing effective vaccination schedules and routes to enhance immunity against ND in layer chickens.

Host genetics drives differences in cecal microbiota composition and immune traits of laying hens raised in the same environment

Vaccination is one of the most effective strategies for preventing infectious diseases but individual vaccine responses are highly heterogeneous. Host genetics and gut microbiota composition are 2 likely drivers of this heterogeneity. We studied 94 animals belonging to 4 lines of laying hens: a White Leghorn experimental line genetically selected for a high antibody response against the Newcastle Disease Virus (NDV) vaccine (ND3) and its unselected control line (CTR), and 2 commercial lines (White Leghorn [LEG] and Rhode Island Red [RIR]). Animals were reared in the same conditions from hatching to 42 d of age, and animals from different genetic lines were mixed. Animals were vaccinated at 22 d of age and their humoral vaccine response against NDV was assessed by hemagglutination inhibition assay and ELISA from blood samples collected at 15, 19, and 21 d after vaccination. The immune parameters studied were the 3 immunoglobulins subtypes A, M, and Y and the blood cell composition was assessed by flow cytometry. The composition of the cecal microbiota was assessed at the end of the experiment by analyzing amplified 16S rRNA gene sequences to obtain amplicon sequence variants (ASV). The 4 lines showed significantly different levels of NDV vaccine response at the 3 measured points, with, logically, a higher response of the genetically selected ND3 line, and intermediate and low responses for the unselected CTR control line and for the 2 commercial lines, respectively. The ND3 line displayed also a higher proportion of immunoglobulins (IgA, IgM, and IgY). The RIR line showed the most different blood cell composition. The 4 lines showed significantly different microbiota characteristics: composition, abundances at all taxonomic levels, and correlations between genera and vaccine response. The tested genetic lines differ for immune parameters and gut microbiota composition and functions. These phenotypic differences can be attributed to genetic differences between lines. Causal relationships between both types of parameters are discussed and will be investigated in further studies.

Newcastle disease virus suppresses antigen presentation via inhibiting IL-12 expression in dendritic cells.

As a potential vectored vaccine, Newcastle disease virus (NDV) has been subject to various studies for vaccine development, while relatively little research has outlined the immunomodulatory effect of the virus in antigen presentation. To elucidate the key inhibitory factor in regulating the interaction of infected dendritic cells (DCs) and T cells, DCs were pretreated with the NDV vaccine strain LaSota as an inhibitor and stimulated with lipopolysaccharide (LPS) for further detection by enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunoblotting, and quantitative real-time polymerase chain reaction (qRT-PCR). The results revealed that NDV infection resulted in the inhibition of interleukin (IL)-12p40 in DCs through a p38 mitogen-activated protein kinase (MAPK)‍-dependent manner, thus inhibiting the synthesis of IL-12p70, leading to the reduction in T cell proliferation and the secretion of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-6 induced by DCs. Consequently, downregulated cytokines accelerated the infection and viral transmission from DCs to T cells. Furthermore, several other strains of NDV also exhibited inhibitory activity. The current study reveals that NDV can modulate the intensity of the innate‍‒‍adaptive immune cell crosstalk critically toward viral invasion improvement, highlighting a novel mechanism of virus-induced immunosuppression and providing new perspectives on the improvement of NDV-vectored vaccine.

Stress-Induced Immunosuppression Inhibits Regional Immune Responses in Chicken Adipose Tissue Partially through Suppressing T Cells by Up-Regulating Steroid Metabolism.

Lipid metabolism plays an important role in maintaining lipid homeostasis and regulating immune functions. However, the regulations and mechanisms of lipid metabolism on the regional immune function of avian adipose tissue (AT) have not been reported. In this study, qRT-PCR was used to investigate the changes and relationships of different lipid metabolism pathways in chicken AT during stress-induced immunosuppression (SIIS) inhibiting immune response to Newcastle disease virus vaccine, then the miRNA regulation patterns of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) gene and its potential applications were further identified. The results showed that AT actively responded to SIIS, and ATGL, CPT1A and HMGCR were all the key genes involved in the processes of SIIS inhibiting the immune responses. SIIS significantly inhibited the natural and specific immune phases of the primary immune response and the initiation phase of the secondary immune response in AT by suppressing T cells by up-regulating steroid anabolism. Moreover, steroid metabolism could play dual roles in regulating the regional immune functions of AT. The miR-29a/c-3p-HMGCR network was a potential regulation mechanism of steroid metabolism in AT, and serum circulating miR-29a/c-3p had the potential as molecular markers. The study can provide valuable references for an in-depth investigation of the regional immune functions regulated by lipid metabolism in AT.

Experimental trials to assess the immune modulatory influence of thyme and ginseng oil on NDV-vaccinated broiler chickens.

The use of traditional medicine against viral diseases in animal production has been practiced worldwide. Herbal extracts possess organic substances that would improve chicken body performance. The current study was designed to evaluate the effect of either thyme or ginseng oil in regard to their immune-modulatory, antiviral, and growth promoter properties. Two hundred and forty-one-day-old broiler chicks were allocated into eight equal groups as the following: group 1; nonvaccinated and nontreated and group 2; Newcastle disease virus (NDV) vaccinated and nontreated. Birds of groups 3 and 4 were treated with thyme oil (200 mg/l of drinking water for 12 hours/day) without or with NDV vaccination. Birds of groups 5 and 6 were treated with ginseng oil (200 mg/l of drinking water for 12 hours/day) without or with NDV vaccination. Birds of groups 7 and 8 were treated with a combination of ginseng oil (100 mg/l of drinking water) and thyme oil (100 mg/l of drinking water) for 12 hours/day. On the 35th day of life, birds in all the experimental groups were given 0.1 ml of a virulent genotype VIId NDV strain suspension containing 106.3 EID50/ml intramuscularly. Administration of ginseng and thyme oils each alone or simultaneously to birds either vaccinated or nonvaccinated elicited a significant improvement in body performance parameters. Administration of thyme and ginseng each alone or concurrently to vaccinated birds (Gp 4, 6, and 8) induced a higher hemagglutination inhibition (HI) titer of 6, 7.3, and 6.3 log2 at 21 days of age, 6.7, 7.6, and 7 log2, at 28 days of age and 7, 8, and 6.8 log2 at 35 days of age, respectively. Challenge with vNDV genotype VII led to an increase in the NDV-specific HI-Ab titers 10 days post challenge in all the experimental groups. In addition, thyme, ginseng oils, or a combination of them improved the protection from mortality in vaccinated birds; by 100%, 100%, and 90%, respectively, compared with 80% protection from mortality in vaccinated-only birds post-NDV challenge. Moreover, NDV-vaccinated birds treated either with thyme; ginseng or their combination showed negative detection of the virus in both tracheal and cloacal swabs and nonvaccinated groups that received oils showed improvement in vNDV shedding in tracheal and cloacal swabs. It could be concluded that the administration of thyme and ginseng essential oils to broilers can improve productive performance parameters, stimulate humoral immunity against, and protect from vNDV infection.

Molecular Identification and Phylogenetic Study Based on the Fusion Gene of Newcastle Disease Virus Isolated from Broiler Poultry Farms in Markazi Province, Iran

Newcastle disease (ND) is an economically significant and extremely spreadable viral illness affecting a wide variety of avian species. ND can rapidly spread within poultry farms and result in considerable economic losses for the global poultry industry. This disease is endemic in Iran, and despite intensive vaccination efforts in the poultry industry, outbreaks of ND occur unexpectedly. This study aimed to isolate the Newcastle disease virus (NDV) from poultry farms with breathing problems in Markazi province, Iran, and investigate the evolutionary relationship and molecular characteristics of the isolates during 2017-2019. To this end, tissue samples (lung, brain, and trachea) were taken from 42 broiler farms exhibiting respiratory symptoms. The samples were inoculated into 9-11-day-old embryonated eggs, and the virus was isolated from 20 (47.6%) of the 42 farms. Subsequently, RT-PCR was used to amplify partial fusion gene sequences from the new isolates. The amplified products were sequenced and compared phylogenetically to the standard pilot dataset (125 selected sequences) generated by the NDV consortium. As determined by phylogenetic analysis, all nine isolates belonged to subgenotype VII.1.1 of genotype VII and were highly similar to isolates from other parts of Iran and China. Moreover, all isolates possessed a polybasic cleavage site motif (112RRQKRF117), characteristic of virulent strains. Furthermore, the present isolates shared a high nucleotide identity (96%) with viruses previously isolated from other provinces of Iran, as determined by BLAST searches and multiple alignments. In addition, they shared a high degree of sequence similarity but were distinct from the existing NDV vaccines. Therefore, the genetic dissimilarity between current vaccine strains and circulating NDVs must be considered in vaccination programs.

The effects of the LaSota strain of oncolytic Newcastle disease virus vaccine on cervical intraepithelial neoplasia Patients-Clinical cohort study

BackgroundCervical cancer is one of the most common malignancies in women, and its treatment has many side effects. Therefore, in this research, the effects of the LaSota strain of oncolytic Newcastle disease virus vaccine on cervical intraepithelial neoplasia (CIN) patients were investigated. Methods15 patients who met the inclusion criteria and diagnosed as CIN II and CIN III were included in the study. The vaccine was injected inside the cervix (neoplasia site) at increasing doses during 21 days, and they were evaluated for adverse events. NDV antibody titer was measured in 90 days and the levels of ki-67 and p16 proteins were studied by immunohistochemistry. Also, the levels of some important inflammatory cytokines in the serum of CIN patients were measured and finally the patients were evaluated according to the final outcomes and the reduction of tumor lesions. ResultsOnly in the first dose of vaccine some patients showed flu-like symptoms. The accumulation of NDV antibodies started on the 7th day of the study and increased until the 90th day. Administration of LaSota vaccine had no significant effect on the expressions of Ki-67 and p16 proteins. Nevertheless, a decrease in the serum levels of Il-1β was observed in patients after the administration of the vaccine, but the serum levels of both Il-2 and INF-γ upregulated significantly. Also, vaccine administration had no significant effect in reducing CIN grades and lesions. ConclusionsIn general, we concluded that LaSota strain of NDV vaccine has no therapeutic effectiveness in CIN patients.

Assessment of Commercial Bivalent Inactivated Newcastle Disease Virus and Infectious Bronchitis Virus Vaccines against Prevalent Isolates in Egypt

Assessment of Commercial Bivalent Inactivated Newcastle Disease Virus and Infectious Bronchitis Virus Vaccines against Prevalent Isolates in Egypt

Superior Efficacy of Apathogenic Genotype I (V4) over Lentogenic Genotype II (LaSota) Live Vaccines against Newcastle Disease Virus Genotype VII.1.1 in Pathogen-Associated Molecular Pattern-H9N2 Vaccinated Broiler Chickens.

A comparison of the efficacy of apathogenic genotype I (V4) and lentogenic genotype II (LaSota) strains of live Newcastle disease virus (NDV) vaccines was performed following vaccination with pathogen-associated molecular pattern (PAMP) H9N2 avian influenza vaccine and challenge with velogenic NDV genotype VII.1.1 (vNDV-VII.1.1). Eight groups (Gs) of day-old chicks were used (n = 25). Groups 1-4 received a single dose of PAMP-H9N2 subcutaneously, while Gs (1, 5) and (2, 6) received eye drops of V4 and LaSota, respectively, as two doses. All Gs, except for 4 and 8, were intramuscularly challenged with vNDV-VII.1.1 at 28 days of age. No signs were detected in Gs 1, 5, 4, and 8. The mortality rates were 0% in Gs 1, 4, 5, and 8; 40% in G2; 46.66% in G6; and 100% in Gs 3 and 7. Lesions were recorded as minimal in Gs 1 and 5, but mild to moderate in Gs 2 and 6. The lowest significant viral shedding was detected in Gs 1, 2, and 5. In conclusion, two successive vaccinations of broilers with a live V4 NDV vaccine provided higher protection against vNDV-VII.1.1 challenge than LaSota. PAMP-H9N2 with live NDV vaccines induced more protection than the live vaccine alone.

Metagenomic characterization reveals virus coinfections associated with Newcastle disease virus among poultry in Kenya.

Newcastle disease (ND) is an endemic viral disease affecting poultry and causing massive economic losses. This cross-sectional purposive study detected coinfections that are associated with the Newcastle disease virus among poultry from selected regions in Kenya. Cloacal (n = 599) and oral-pharyngeal (n = 435) swab samples were collected and pooled into 17 and 15 samples, respectively. A total of 17,034,948 and 7,751,974 paired-end reads with an average of 200 nucleotides were generated from the cloacal and oral-pharyngeal swab samples, respectively. Analysis of the de novo assembled contigs identified 177 and 18 cloacal and oral-pharyngeal contigs, respectively with hits to viral sequences, as determined by BLASTx and BLASTn analyses. Several known and unknown representatives of Coronaviridae, Picobirnaviridae, Reoviridae, Retroviridae, and unclassified Deltavirus were identified in the cloacal swab samples. However, no Newcastle disease virus (family Paramyxoviridae) was detected in the cloacal swabs, although they were detected in the oropharyngeal swabs of chickens sampled in Nairobi, Busia, and Trans Nzoia. Additionally, sequences representative of Paramyxoviridae, Coronaviridae, and Retroviridae were identified in the oral-pharyngeal swab samples. Infectious bronchitis virus and rotavirus were chickens' most prevalent coinfections associated with the Newcastle disease virus. The detection of these coinfections suggests that these viruses are significant threats to the control of Newcastle disease as the Newcastle disease virus vaccines are known to fail because of these coinfections. Therefore, this study provides important information that will help improve disease diagnosis and vaccine development for coinfections associated with the Newcastle disease virus.

Identification and evaluation in-vitro of conserved peptides with high affinity to MHC-I as potential protective epitopes for Newcastle disease virus vaccines

BackgroundNewcastle disease (ND) is a major threat to the poultry industry, leading to significant economic losses. The current ND vaccines, usually based on active or attenuated strains, are only partially effective and can cause adverse effects post-vaccination. Therefore, the development of safer and more efficient vaccines is necessary. Epitopes represent the antigenic portion of the pathogen and their identification and use for immunization could lead to safer and more effective vaccines. However, the prediction of protective epitopes for a pathogen is a major challenge, especially taking into account the immune system of the target species.ResultsIn this study, we utilized an artificial intelligence algorithm to predict ND virus (NDV) peptides that exhibit high affinity to the chicken MHC-I complex. We selected the peptides that are conserved across different NDV genotypes and absent in the chicken proteome. From the filtered peptides, we synthesized the five peptides with the highest affinities for the L, HN, and F proteins of NDV. We evaluated these peptides in-vitro for their ability to elicit cell-mediated immunity, which was measured by the lymphocyte proliferation in spleen cells of chickens previously immunized with NDV.ConclusionsOur study identified five peptides with high affinity to MHC-I that have the potential to serve as protective epitopes and could be utilized for the development of multi-epitope NDV vaccines. This approach can provide a safer and more efficient method for NDV immunization.

Thermostability and Immunostimulatory Activity of Cationic Liposome-based Newcastle Disease Virus Vaccine

Cold storage is a huge challenge in maintaining vaccine potency especially in low and middle income countries (LMIC). The aim of this research was to investigate the heat and freeze-thaw stability of Newcastle disease virus (NDV) vaccine encapsulated in 1,2-dioleoyl-3-trimethyl ammonium propane (DOTAP) cationic liposomes as well as its immunostimulatory activity. DOTAP cationic liposomes were prepared using the thin-film hydration technique and characterized for morphology, particle size, polydispersity index, zeta potential and encapsulation efficiency. Experimental birds were challenged with Herts 33 velogenic strain of NDV. Haemagglutination inhibition test was used to assess the formation of antibodies in the birds. The cationic liposomes were further stored at 28 oC or subjected to freeze-thaw cycles for 7 weeks and then assessed for stability using the same parameters of particle size, polydispersity index, zeta potential, morphology, antibody titre or differential white blood cell count. Using TEM, the morphology of the liposomes was found to be spherical, and using Malvern Zetasizer, particle size was in the nanosize range. Zetapotential and polydispersity indices were positive and below 0.1. The HI test showed that the DOTAP cationic liposomes stimulated a higher titre of antibodies than the commercial vaccine. Heat and freeze-thaw stability studies after 7 weeks of storage showed slight aggregation and elevated particle sizes and wider distribution. The HI titer was appreciably lower after secondary immunization with the DOTAP cationic liposomes stored at 28 oC but elevated for the product kept at freeze-thaw conditions. The immunized birds were 100 % protected from the virulent strain of NDV. In conclusion, the development of the DOTAP-cationic liposomes encapsulating NDV vaccine administered orally was found to be more stable under freeze-thaw conditions than when it was exposed to heat for 7 weeks. The liposome-based vaccine had more immunostimulatory effects than the commercial sample.

C-terminal truncation of the hemagglutinin-neuraminidase (HN) protein enhances the virulence and immunogenicity of Newcastle disease virus (NDV) vaccine strain V4.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein with receptor recognition ability that plays an important role in the infection of cells by NDV. An alignment of NDV HN protein sequences of different genotypes showed that vaccine strains of NDV, such as the LaSota strain, generally have an HN protein of 577 amino acids. In comparison, the HN protein of the V4 strain has 616 amino acids, with 39 more amino acids at the C-terminus. In this study, we generated a recombinant NDV (rNDV) with a 39-amino-acid truncation at the HN C-terminus based on the full-length cDNA clone of the V4 strain. This rNDV, named rV4-HN-tr, displayed thermostability similar to that of the parental V4 strain. However, growth kinetics and pathogenicity analysis suggested that rV4-HN-tr is more virulent than the V4 strain. Notably, the C-terminus of HN affected the ability of the virus to adsorb onto cells. Structural predictions further suggested that the C-terminus of HN may obstruct the sialic acid binding site. Immunization of chickens with rV4-HN-tr induced a 3.5-fold higher level of NDV-specific antibodies than that obtained with the V4 strain and provided 100% immune protection against NDV challenge. Our study suggests that rV4-HN-tr is a thermostable, safe, and highly efficient vaccine candidate against Newcastle disease.

Immunity Evaluation of Inactivated Newcastle Disease Virus Vaccine Inoculated at Different Doses in Day-old Specific-Pathogen-Free Chicks

This research aims to evaluate the immunogenicity of different doses of HIPRAVIAR® BPL2 inactivated Newcastle disease virus (NDV) LaSota vaccine. Specific-pathogen-free day-old chicks were divided into 3 different groups, and each group was vaccinated subcutaneously with the vaccine dose of 0.1, 0.2, and 0.5 ml, respectively. Blood samples were collected to measure NDV-specific antibody titers using a hemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA). The HI result showed that birds vaccinated with 0.5 ml HIPRAVIAR® BPL2 vaccine showed an increased statistically significant antibody titer compared to the other doses. Similarly, the ELISA result corroborated the HI finding. No significant difference between the results was detected when the antibody titers were measured using two ELISA kits, Biocheck CK116, and CIVTEST® AVI NDV. The percentage antibody-positive test based on HI amongst the different days post-vaccination showed that all the birds were positive from 28 to 42 days following vaccination with HIPRAVIAR® BPL2 0.5 ml (group D), whereas the highest percentage of antibody positivity were 80% and 70% at 42 days post-vaccination with HIPRAVIAR® BPL2 0.1 ml (group B) and HIPRAVIAR® BPL2 0.2 ml (group C), respectively. In conclusion, besides the difference in seroconversion, all the vaccine doses used had important levels of seroconversion and positivity.

A Booster with a Genotype-Matched Inactivated Newcastle Disease Virus (NDV) Vaccine Candidate Provides Better Protection against a Virulent Genotype XIII.2 Virus.

Newcastle disease (ND) is endemic in Bangladesh. Locally produced or imported live Newcastle disease virus (NDV) vaccines based on lentogenic virus strains, locally produced live vaccines of the mesogenic Mukteswar strain, as well as imported inactivated vaccines of lentogenic strains, are being used in Bangladesh under different vaccination regimens. Despite these vaccinations, frequent outbreaks of ND are being reported in Bangladesh. Here we compared the efficacy of booster immunization with three different vaccines in chickens that had been primed with two doses of live LaSota vaccine. A total of 30 birds (Group A) were primed with two doses of live LaSota virus (genotype II) vaccine at days 7 and 28, while 20 birds (Group B) remained unvaccinated. At day 60, birds of Group A were divided into three sub-groups, which received booster immunizations with three different vaccines; A1: live LaSota vaccine, A2: inactivated LaSota vaccine, and A3: inactivated genotype XIII.2 vaccine (BD-C161/2010 strain from Bangladesh). Two weeks after booster vaccination (at day 74), all vaccinated birds (A1-A3) and half of the unvaccinated birds (B1) were challenged with a genotype XIII.2 virulent NDV (BD-C161/2010). A moderate antibody response was observed after the primary vaccination, which substantially increased after the booster vaccination in all groups. The mean HI titers induced by the inactivated LaSota vaccine (8.0 log2/5.0 log2 with LaSota/BD-C161/2010 HI antigen) and the inactivated BD-C161/2010 vaccine (6.7 log2/6.2 log2 with LaSota/BD-C161/2010 HI antigen) were significantly higher than those induced by the LaSota live booster vaccine (3.6 log2/2.6 log2 with LaSota/BD-C161/2010 HI antigen). Despite the differences in the antibody titers, all chickens (A1-A3) survived the virulent NDV challenge, while all the unvaccinated challenged birds died. Among the vaccinated groups, however, 50% of the chickens in Group A1 (live LaSota booster immunization) shed virus at 5- and 7-days post challenge (dpc), while 20% and 10% of the chickens in Group A2 (inactivated LaSota booster immunization) shed virus at 3 and 5 dpc, respectively, and only one chicken (10%) in Group A3 shed virus at 5 dpc. In conclusion, the genotype-matched inactivated NDV booster vaccine offers complete clinical protection and a significant reduction in virus shedding.

Identification of Embryonic Chicken Proteases Activating Newcastle Disease Virus and Their Roles in the Pathogenicity of Virus Used as In Ovo Vaccine.

In ovo vaccination is an attractive immunization approach for chickens. However, most live Newcastle disease virus (NDV) vaccine strains used safely after hatching are unsafe as in ovo vaccines due to their high pathogenicity for chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. Our previous studies reported that NDV strain TS09-C was a safe in ovo vaccine, and the F protein cleavage site (FCS) containing three basic amino acids (3B-FCS) was the crucial determinant of the attenuation of TS09-C in chicken embryos. Here, five trypsin-like proteases that activated NDV in chicken embryos were identified. The F protein with 3B-FCS was sensitive to the proteases Tmprss4, Tmprss9, and F7, was present in fewer tissue cells of chicken embryos, which limited the viral tropism, and was responsible for the attenuation of NDV with 3B-FCS, while the F protein with FCS containing two basic amino acids could be cleaved not only by Tmprss4, Tmprss9, and F7 but also by Prss23 and Cfd, was present in most tissue cells, and thereby was responsible for broad tissue tropism and high pathogenicity of virus in chicken embryos. Furthermore, when mixed with the protease inhibitors aprotinin and camostat, NDV with 2B-FCS exhibited greatly weakened pathogenicity in chicken embryos. Thus, our results extend the understanding of the molecular mechanism of NDV pathogenicity in chicken embryos and provide a novel molecular target for the rational design of in ovo vaccines, ensuring uniform and effective vaccine delivery and earlier induction of immune protection by the time of hatching. IMPORTANCE As an attractive immunization approach for chickens, in ovo vaccination can induce a considerable degree of protection by the time of hatching, provide support in closing the window in which birds are susceptible to infection, facilitate fast and uniform vaccine delivery, and reduce labor costs by the use of mechanized injectors. The commercial live Newcastle disease virus (NDV) vaccine strains are not safe for in ovo vaccination and cause the death of chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. In the present study, we identified five trypsin-like proteases that activate NDV in chicken embryos and elucidated their roles in the tissue tropism and pathogenicity of NDV used as in ovo vaccine. Finally, we revealed the molecular basis for the pathogenicity of NDV in chicken embryos and provided a novel strategy for the rational design of in ovo ND vaccines.

Monitoring of Newcastle Disease Virus Vaccine Strain Replication in Embryonated Chicken Eggs by Reverse Transcription-Polymerase Chain Reaction.

The knowledge of virus and replication kinetics plays a key role in developing a vaccine. This study aimed to monitor the replication process and determine the best harvesting time of the Newcastle disease virus (NDV) V4 vaccine strain in the allantoic fluids of specific pathogen-free (SPF)-embryonated chicken eggs (ECEs) by reverse transcription-polymerase chain reaction (RT-PCR), hemagglutination (HA), and egg infective dose 50% (EID50) tests. For this purpose, the V4 vaccine strain of the virus was intra-allantoically inoculated into 96 10-day-old SPF-ECEs at the rate of 0.1 mL/ECE. The allantoic fluids of the inoculated eggs were collected from six eggs at six-hour intervals up to 96 hours post-infection (hpi). The harvested suspensions were confirmed to contain the NDV by the mentioned serologic and molecular techniques. Based on the results, the virus was first detected at 36 hpi in ECEs by RT-PCR. The peak of HA and EID50 titers in allantoic fluids started at 42 hpi, and the titers were at the highest level until the end of the experiment. The results indicated that the best virus harvesting time for the NDV V4 vaccine strain in ECEs is between 42-60 hpi. These findings pave the way for adequate and enhanced production rate, immunogenicity, and cost-related parameters in the V4 Newcastle vaccine development.

Immunogenicity and protective efficacy of an inactivated Newcastle disease virus vaccine encapsulated in poly-(lactic-co-glycolic acid) nanoparticles

An immunization experiment was conducted in specific pathogen-free chickens with the inactivated Newcastle disease virus (NDV) vaccine encapsulated in the poly-(lactic-co-glycolic) acid (PLGA) nanoparticles (NP) to evaluate its immunogenicity and protective efficacy. The NDV vaccine was prepared by inactivating one virulent Indian strain of NDV belonging to Genotype VII by using beta-propiolactone. PLGA nanoparticles encapsulating inactivated NDV were prepared by the solvent evaporation method. Scanning electron microscopy and zeta sizer analysis revealed that the (PLGA+NDV) NP were spherical, with an average size of 300 nm, having a zeta potential of -6 mV. The encapsulation efficiency and loading efficiency were 72% and 2.4%, respectively. On immunization trial in chicken, the (PLGA+NDV) NP induced significantly (P < 0.0001) higher levels of HI and IgY antibodies with the peak HI titer of 28 and higher expression of IL-4 mRNA. The consistency of higher antibody levels suggests slow and pulsatile release of the antigens from the (PLGA+NDV) NP. The nano-NDV vaccine also induced cell mediated immunity with higher expression of IFN-γ indicating strong Th1 mediated immune responses in contrast to the commercial oil adjuvanted inactivated NDV vaccine. Further, the (PLGA+NDV) NP afforded 100% protection against the virulent NDV challenge. Our results suggested that PLGA NP have adjuvant potential on induction of humoral as well as Th1 biased cell mediated immune responses and also enhanced protective efficacy of the inactivated NDV vaccine. This study provides an insight for development of PLGA NP based inactivated NDV vaccine using the same genotype circulating in the field as well as for other avian diseases at exigencies.