Genotype VII Newcastle Disease Virus Research Articles

Evaluation of the Newcastle disease virus genotype VII–mismatched vaccines in SPF chickens: A challenge efficacy study

Newcastle disease virus (NDV) strains, while falling under a single serotype, are classified into distinct genotypes. Genotype VII virulent NDVs pose a significant threat to poultry due to their association with high mortality rates and economic losses. This study aimed to evaluate the efficacy of three commercial live vaccines based on genotype II against genotype VII virulent NDV (vNDV) in specific pathogen-free (SPF) chickens. Forty one-day-old chickens were randomly divided into four groups (n = 10) and inoculated with one dose of each ND pneumotropic vaccine—B1, Clone.12IR, and La Sota—or received phosphate-buffered saline (PBS) as a control at eight days of age via eye drop. At 28 days of age (20th post-vaccination days), chickens were intramuscularly challenged with genotype VII virulent NDV (≥ 105 LD50). Serum samples were collected at 28 days of age (challenge day), 7 and 14 post-challenge days to measure NDV antibodies via the hemagglutination inhibition (HI) test. Cloacal and oropharyngeal swabs were taken on the 3rd, 5th, 7th, and 10th post-challenge days to evaluate virus shedding. Vaccinated groups exhibited significantly higher antibody titers and greater protection levels compared to the control group (P≤ 0.001). While HI antibody titer was not different at 28 and 35 days of age between vaccinated chickens, the Clone.12IR groups showed higher HI antibody titer compared to B1 at day 42 of age (9.43 vs. 7.42; P≤ 0.002). La Sota and Clone.12IR vaccines demonstrated superior protection against mortality compared to the B1 vaccine (90 %, 80% vs. 60 %, respectively) with 6.0 and 2.67 odds ratio of survivability. All three mismatched vaccines effectively curbed the shedding of virulent genotype VII NDV, with 0 % to 11 % positive cloacal samples up to the 3rd post-challenge day. These findings demonstrate that in the experimental setting, the administration of mismatched ND vaccines, particularly La Sota and Clone.12IR, confer protection against genotype VII virulent NDV and control viral shedding, which can help to develop effective vaccination strategies to mitigate the impact of vNDV outbreaks in the poultry farms.

Comparison of HVT-ND recombinant and convection-based Newcastle disease vaccination programs in the protection against the genotype VII NDV challenges: an experimental study.

Newcastle disease virus (NDV) belongs to the Avulavirus genus and Paramyxoviridae family virus that causes acute, highly infectious Newcastle disease in poultry. The two proteins of haemagglutinin neuraminidase (HN) and fusion (F) are key virulence factors with an important role in its immunogenicity. Genotype VII NDV is still amongthe most serious viral hazards to the poultry industry worldwide. In this study, a commercial vector vaccine (HVT-NDV) was evaluated compared to the conventional vaccination strategy against Iranian genotype VII. This experiment showed that the group receiving the conventional vaccination strategy had higher antibodies, fewer clinical signs, and lower viral loads in tracheal swabs and feces. Also, two vaccine groups showed significant difference, which could have resulted from two extra vaccine doses in the conventional group. However, except for antibody levels in commercial chickens in the Iran new-generation vaccine, this difference was minor. Further, both groups showed 100% protection in the challenge study. Despite the phylogenetic gap between the NDV-F gene placed in the vector vaccine and the challenge virus (genotypes I and VII, respectively), the rHVT-NDV vaccine offered strong clinical protection and decreased challenge virus shedding considerably.

Protective effectiveness of two vaccination schemes against the prevalent Egyptian strain of Newcastle disease virus genotype VII.

Despite the strict preventive immunization used in Egypt, Newcastle disease remained a prospective risk to the commercial and backyard chicken industries. The severe economic losses caused by the Newcastle disease virus (NDV) highlight the importance of the trials for the improvement and development of vaccines and vaccination programs. In the present study, we evaluated the effectiveness of two vaccination schemes for protection against the velogenic NDV (vNDV) challenge. Four groups (A-D) of commercial broiler chickens were used. Two groups (G-A and G-B) were vaccinated with priming live HB1 GII simultaneously with inactivated GVII vaccines at 5 days of age, then boosted with live LaSota GII vaccine in group A and live recombinant NDV GVII vaccine in group B on day 16. Groups A to C were challenged with NDV/Chicken/Egypt/ALEX/ZU-NM99/2019 strain (106 Embryo infective dose 50/0.1 ml) at 28 days of age. Two vaccination schemes achieved 93.3% clinical protection against NDV with body gain enhancement; whereas, 80% of the unvaccinated-challenged birds died. On day 28, the mean HI antibody titers were 4.3 ± 0.33 and 5.3 ± 0.33 log2 in groups A and B, respectively. As well as both programs remarkably reduced virus shedding. The two vaccination schemes displayed close protection efficacy against the vNDV challenge. Therefore, using the combination of a live attenuated vaccine with an inactivated genetically matched strain vaccine and then boosting it with one of the available live vaccines could be considered one of the most effective programs against current field vNDV infection in Egypt.

Evaluation of inactivated avian influenza virus and Newcastle disease virus bivalent vaccination program against newly circulated H5N8 and NDV strains

Avian influenza virus (AIV) and Newcastle disease virus (NDV) are respiratory illness syndromes that have recently been detected in vaccinated flocks and are causing major financial losses in the chicken farming industry. The objective was to evaluate the efficacy of Valley Vac H5Plus NDVg7 vaccine in protecting chickens against the H5N8 and NDV strains that have recently been circulating in comparison with the efficacy of the commercially available bivalent H5+ND7 vaccine. In contrast to the H5+ND7 vaccine, which was made of genetically distinct H5N8/2018 clade 2.3.4.4b genotype group (G5), H9N2/2016, H5N1/2017, and genetically comparable NDV genotype VII 1.1/2019 of the recently circulating challenge viruses, the Valley Vac H5Plus NDVg7 vaccine consisted of the recently isolated (RG HPAI H5N1 AIV/2015 Clade 2.2.1.2, RG HPAIV H5N8/2020 Clade 2.3.4.4b genotype group 6 (G6), and NDV genotype VII 1.1/2012) which were genetically similar to challenged strains. To determine the effectiveness of the Valley Vac H5Plus NDVg7 vaccine, a total of 70-day-old commercial chicks were divided into 7 groups of 10 birds each. Groups (G1 and G4) received Valley Vac H5Plus NDVg7 vaccine. Groups (G2 and G5) groups received commercial H5+ND7 vaccine. While groups (G3 and G6) were kept nonvaccinated, and group (G7) was kept as a nonchallenged and nonvaccinated. After 3-wk post vaccination (WPV), groups G1, G2, and G3 were challenged with A/Duck/ Egypt/SMG4/2019(H5N8) genotype G6. On the other hand, groups G4, G5, G6 were challenged with NDV/EGYPT/18629F/2018 genotype VII 1.1 with an intranasal injection of 0.1 mL. Antibody titer was calculated at the first 3 wk after vaccination, and the viral shedding titer was calculated at 3-, 5-, and 7-days post challenge. Mortality and morbidity rates were monitored daily during the experiment, and for the first 10 d after the challenge, to provide an estimate of the protection rate. The results showed that a single dosage of 0.5 mL per bird of Valley Vac H5Plus NDVg7 vaccine provides 80% protection against both H5N8 and NDV, compared to the bivalent H5+ND7 vaccine, which provided 20 and 80% protection against H5N8 and NDV, respectively. In addition, 0.5 mL per bird of Valley Vac H5Plus NDVg7 vaccine produced a greater immune response against both viruses than commercial vaccination at 1 to 3 WPV with a significant difference at 1 WPV for H5N8 and a comparatively higher immune response for NDV. Furthermore, it reduced virus shedding of H5N8 on the third, fifth, seventh, and tenth days lower than H5+ND7 vaccine with a significant difference on the third day for H5N8 and relatively lower than bivalent H5+ND7 vaccine for NDV with a significant difference on the fifth day. The Valley vaccinated group demonstrated more tissue intactness compared to the commercially vaccinated group against the H5N8 challenge, however the bivalent commercially vaccinated group showed the similar level of tissue integrity against NDV. In conclusion, Valley Vac H5Plus NDVg7 that contains the genetically similar strain to recently circulating challenged virus (H5N8 genotype G6) provided better protection with greater immune response and decreased the amount of virus shed against H5N8 genotype G6 and showed less histopathological alteration than the commercial bivalent H5+ND7 vaccine that contain genetically distinct (H5N8 genotype G5). However the Valley Vac H5Plus NDVg7 provided the same protection with relatively high immune response and relatively decreased the amount of virus shed and showed equal tissue integrity than the commercial bivalent H5+ND7 vaccine against NDV genotype VII 1.1 that contain the same genotype of NDV genotype VII 1.1.

Protection against genotype VII Newcastle disease virus by a mucosal subunit vaccination based on bacterium-like particles bearing the F or HN antigen

Genotype VII Newcastle disease viruses (NDV) are still epidemic in many countries in chicken and waterfowl despite intensive vaccination with conventional live and inactivated vaccines. Here, we developed an effective mucosal subunit vaccine based on a bacterium-like particles (BLPs) delivery platform derived from Lactococcus lactis. The NDV protective antigen F or HN fused protein anchor (PA) was expressed by recombinant baculovirus and loaded on the surface of BLPs, resulting in BLPs-F and BLPs-HN, respectively. Efficient uptake of BLPs-F/HN by antigen-presenting cells activated the innate immune system depending mainly on the combination of chicken TLR2 type 1 (chTLR2t1) and chicken TLR1 type 1 (chTLR1t1) was observed. Delivered intranasally, BLPs-F, BLPs-HN, or BLPs-F/HN (a mixture containing equal amounts of BLPs-F and BLPs-HN) elicited robust local NDV-specific SIgA in the trachea as well as systemic neutralizing antibody and a mixed Th1/Th2 immune response in chickens. Notably, BLPs-F/HN provided as high as 90 % protection rate against intranasal challenge with a lethal dose of virulent genotype VII NDV NA-1 strain. These data indicate that this BLP-based subunit vaccine has the potential to be a novel mucosal vaccine against genotype VII NDV infection.

Simultaneous Expression of Chicken Granulocyte Monocyte Colony-Stimulating Factor and the Hemagglutinin-Neuraminidase Epitope of the Virulent Newcastle Disease Virus Genotype VII C22 Strain in a Functional Synthetic Recombinant Adenovirus as a Genotype-Matched Vaccine with Potential Antiviral Activity.

When it comes to the prevention of clinical signs and mortality associated with infection of the Newcastle disease virus (NDV), vaccination has been very effective. However, recent evidence has proven that more highly virulent strains are emerging that bypass existing immune protection and pose a serious threat to the global poultry industry. Here, a novel rescued adenovirus 5-coexpressed chicken granulocyte monocyte colony-stimulating factor (ChGM-CSF) bio-adjuvant and C22-hemagglutinin-neuraminidase (HN) boosted chickens' immunological genetic resistance and thus improved the immunological effectiveness of the critical new-generation vaccine in vitro and in vivo. Accordingly, the hemagglutination inhibition (HI) titers (log2) of the recombinant adenovirus (rAdv)-ChGM-CSF-HN-immunized chickens had greater, more persistent, and longer-lasting NDV-specific antibodies than the La Sota and rAdv-HN-inoculated birds. Moreover, humoral and adaptive immunological conditions were shown to be in harmony after rAdv-ChGM-CSF-HN inoculation and uniformly enhanced the expression of alpha interferon (IFN-α), IFN-β, IFN-γ, interleukin-1β (IL-1β), IL-2, IL-16, IL-18, and IL-22. Postchallenge, the control challenge (CC), wild-type adenovirus (wtAdv), and rAdv-ChGM-CSF groups developed unique NDV clinical manifestations, significant viral shedding, high tissue viral loads, gross and microscopic lesions, and 100% mortality within 7 days. The La Sota, rAdv-HN, and rAdv-ChGM-CSF-HN groups were healthy and had 100% survival rates. The rAdv-ChGM-CSF-HN group swiftly regulated and stopped viral shedding and had lower tissue viral loads than all groups at 5 days postchallenge (dpc). Thus, the antiviral activity of ChGM-CSF offered robust immune protection in the face of challenge and reduced viral replication convincingly. Our advance innovation concepts, combining ChGM-CSF with a field-circulating strain epitope, could lead to the development of a safe, genotype-matched, universal transgenic vaccine that could eradicate the disease globally, reducing poverty and food insecurity. IMPORTANCE We studied the biological characterization of the developed functional synthetic recombinant adenoviruses, which showed a high degree of safety, thermostability, and genetic stability for up to 20 passages. It was demonstrated through both in vitro and in vivo testing that the immunogenicity of the proposed vaccine, which uses the T2A peptide from the Thosea asigna virus capsid protein supported by glycine and serine, helps with efficiency to generate a multicistronic vector, enables expression of two functional proteins in rAdv-ChGM-CSF-HN, and is superior to that of comparable vaccines. Additionally, adenovirus can be used to produce vaccines matching the virulent field-circulating strain epitope. Because there is no preexisting human adenoviral immunity detected in animals, the potency of adenoviral vaccines looks promising. Also, it ensures that the living vector does not carry the resistance gene that codes for the kanamycin antibiotic. Accordingly, a human recombinant adenoviral vaccine that has undergone biological improvements is beneficial and important.

EVALUATION OF DIFFERENT NEWCASTLE DISEASE VIRUS VACCINATION REGIMES AGAINST CHALLENGE WITH RECENTLY ISOLATED GENOTYPE VII VIRUS FROM EGYPT

Newcastle disease virus (NDV) genotype VII is incriminated in the currently circulating NDV outbreaks in the Middle East region. In this study, evaluation of different vaccination regimes including genetically-matched or mismatched vaccines to the currently circulating field virulent NDV (vNDV) genotype VII was performed. One-day-old Arbor Acres broiler chicks were divided into nine groups; groups 1 to 3 were vaccinated with live or inactivated genetic-mismatched vaccines (genotype II) or both of them. Groups 4 to 6 were vaccinated with either live or inactivated genetic-matched vaccine to vNDV genotype VII or combination of them. Group (Gp) 7 was vaccinated with a combination of inactivated genetic-matched and live genetic-mismatched vaccines to vNDV genotype VII while groups 8 and 9 were kept as control non-vaccinated. The groups that received a combination of live and inactivated vaccines from either genetically-matched or mismatched origins had the highest serological responses and protection against mortality which was 100%. The two groups received a combination of inactivated genetic matched vaccine and live vaccines of either genetic-matched or mismatched origins had the lowest clinical index and were nearly completely protected against vNDV clinical signs. The virus tracheal and cloacal shedding titers and number of shedders were significantly reduced or nearly neglicable in the instance of application of inactivated genetic-matched vaccine to the challenge virus either alone or boosted with live genetic-matched or mismatched vaccine. In consistent inactivated genetic-matched vaccine inhibited the transmissibility of the challenged virus to contacted birds. We concluded from our results that application of NDV vaccination regimes included a combination of inactivated NDV genotype VII vaccine and live vaccine regardless of its genotype provides better clinical protection and minimize virus shedding and subsequently decrease transmissibility and virus load to the surrounding environment.

Enhanced Protection and Blocked Viral Shedding for Broiler Chickens in Challenge with Newcastle Disease Virus Genotype VII by Generation of Oil Inactivated Vaccine Antigenically-Matched to The Endemic Virus in Egypt

Enhanced Protection and Blocked Viral Shedding for Broiler Chickens in Challenge with Newcastle Disease Virus Genotype VII by Generation of Oil Inactivated Vaccine Antigenically-Matched to The Endemic Virus in Egypt

Characterization of Chicken-Derived Genotype VII Newcastle Disease Virus Isolates from Northwest China

Newcastle disease virus (NDV) threatens global poultry production, with genotype VII the most prevalent strain in China. However, little information is available regarding viral multiplication and pathogenicity based inoculation route. The objectives of this study were to sequence NDV VII isolates and to analyze their biological characteristics in detail. A total of 86 oral and cloacal swabs were collected from Shaanxi and Gansu provinces in northwest China. Identification of genotype VII NDV based on the M gene was performed by qPCR. Viral multiplication and pathogenicity were assessed as a function of route of infection. We observed increased morbidity and mortality using intravenous injection, whereas intranasal, intraocular, and cloacal infections resulted in slower progression and milder clinical disease, with viral proliferation obvious in different tissues. These results provide an important basis for the clinical control and prevention of NDV epidemics in poultry.

Construction of Novel Thermostable Chimeric Vaccine Candidates for Genotype VII Newcastle Disease Virus.

Genotype VII Newcastle Disease Virus (NDV) has caused a pandemic in many countries and usually causes fatal consequences in infected chickens. Although current commercial attenuated NDV vaccines can provide an ideal protection against genotype VII NDV, they cannot completely prevent the infection and viral shedding, and the genotype of some vaccine strains cannot match with the prevalent strain. In this study, in order to construct a thermostable and genotype VII-matched live attenuated vaccine, we used a thermostable genotype VIII virulent HR09 strain as the backbone and replaced its F gene with that of the genotype VII DT-2014 strain. Meanwhile, the cleavage site of F gene of DT-2014 was mutated to that of class I F protein and avirulent class II F protein, respectively. The results showed that the two chimeric viruses, designated rcHR09-CI and rcHR09-CII, shared a similar growth kinetics and thermostability with their parental HR09 strain. Mean death time (MDT) and intracerebral pathogenicity index (ICPI) tests showed that the two chimeric viruses were highly attenuated. Though both chimeric NDVs and La Sota vaccine strain could provide complete protection to immunized chickens against the challenge of virulent genotype VII ZJ1 strain, the two chimeric NDVs could induce a higher level of antibody response against ZJ1 strain and could significantly reduce the viral shedding compared with La Sota vaccine strain. In conclusion, our study constructed two chimeric thermostable genotype VII-matched NDV vaccine candidates, which provided complete protection against the challenge of virulent genotype VII NDV.

Expression Profiles of Immune-Related Genes and Apoptosis Study of Avian Intraepithelial-Natural Killer Cells in Chickens Inoculated with Vaccine Strain of Newcastle Disease Virus (NDV) and Challenged with Virulent NDV.

In spite of the available information on the role of natural killer (NK) cells in several viral infections, the interactions between chicken intraepithelial-NK (IEL-NK) cells and Newcastle disease virus (NDV) are poorly understood. In this study, we investigated these interactions following the inoculation of chickens with NDV vaccine strain LaSota and subsequent challenge with velogenic NDV (vNDV) genotype VII (GVII) and VIII (GVIII), through quantification of IEL-NK cell's apoptosis and expression profiling of its surface receptors. Specific-pathogen-free chickens were randomly divided into six groups, as follows: one group of an uninfected control, one group infected with NDV LaSota, two groups each infected with either GVII or GVIII, and two groups inoculated with NDV LaSota and challenged with either GVII (LaSota-genotype VII [LSGVII]) or GVIII (LaSota-genotype VIII [LSGVIII]). Avian intraepithelial lymphocytes (IEL) were isolated from the duodenal loops, and CD3- cells were characterized. Immunophenotyping and apoptosis analysis of CD3-/CD25+/CD45+IEL NK cells were conducted using a flow cytometer. In addition, a gene expression study was conducted using real-time quantitative PCR. Data were analyzed using two-way analysis of variance. The results showed that vNDV GVII or GVIII caused apoptosis of IEL-NK cells; however, following inoculation of LSGVII or LSGVIII, the effect of vNDV GVII and GVIII to cause a reduction in the population of viable IEL-NK cells was significantly reduced. Furthermore, the expression profiles of activating receptors CD69, NK-lysin, and IFN-γ, were generally upregulated in chickens inoculated with LSGVII or LSGVIII. In contrast, B-NK, an inhibitory receptor, was downregulated in these treatment groups. In NDV GVII- and GVIII-challenged groups, however, B-NK was upregulated, whereas the other receptors were generally downregulated. The findings of this study showed that NDV vaccine strain LaSota may prevent apoptosis and cause upregulation of activating receptors of chicken IEL-NK cells in velogenic virus-challenged settings.

In vitro investigation of the antiviral activity of propolis and chitosan nanoparticles against the genotype VII Newcastle disease virus.

The Newcastle disease virus (NDV) is considered a serious threat to global poultry production. Despite the availability of vaccines, it remains a major devastating epidemic responsible for great economic losses. The development of novel virus-controlling strategies is therefore an urgent need. The present study investigated for the first time the antiviral efficacy of propolis and chitosan nanoparticles against two NDV isolates, MW881875 and MW881876, recovered from vaccinated commercial broiler farms in KafrEl Sheikh Governorate, Egypt. The polygenetic analysis focused on the F and M genes, with one isolate having a 97% identity with the genotype VII NDV Israeli strain. On the other hand, the identified isolates showed high genetic variation and only 76% identity with the LaSota vaccine (genotype II). More interestingly, the cell cytotoxic concentrations of chitosan, propolis, and a propolis–chitosan mixture against Vero cells were 327.41 ± 12.63, 109.48 ± 8.36, and 231.78 ± 11.46 μg/ml, respectively. The median tissue culture infectious dose (TCID50) assay demonstrated that the nanoparticles have antiviral effects after NDV exposure resulting in significant decrease in viral titer (TCID50) by 2, 2.66, and 2.5 log10 at 62 μg/ml of chitosan, 13 μg/ml of propolis, and 30 μg/ml of the propolis–chitosan mixture, respectively, compared with the control TCID50 value of 4 log10. Taken together, the results provide novel insights into the potentially promising roles of propolis and chitosan as novel, safe, and effective antiviral agents against NDV.

Molecular Characterization of Recently Classified Newcastle Disease Virus Genotype VII.1.1 Isolated from Egypt

Newcastle disease virus (NDV) is classified as Avian OrthoAvulavirus-1 (AOAV-1) and is still one of the most important poultry diseases in the world. Collected tissue specimens (trachea, cecal tonsils, and spleen) from 85 chicken flocks (broilers, layers, and native breeds) raised in 5 Egyptian governorates (El-Qalubia, El-Behaira, El-Menofia, El-Gharbia, and Alexandria) in the period between March 2019 to February 2020. The flocks had a history of vaccination against ND and showed clinical signs and lesions of infection. We characterized the isolates molecularly using conventional RT-PCR via amplifying the NDV full Fusion protein gene. The analysis of the full F-gene sequence revealed that our isolates were classified as genotype VII with the characteristic amino acids motif in the fusion protein cleavage site for the velogenic NDV (vNDV) strains. Among the molecularly identified NDV Genotype VII isolates, 5 representative isolates in different 5 Egyptian Governorates were sequenced for full fusion protein gene (sequence). The obtained sequences were submitted on GenBank: MW580389, MW580388, MW580387, MW566177, and MW590306. The five isolates' full-length F protein gene amino acid sequences revealed 99.5% identity with the Reference GenBank NDV/Egypt/ch/MN51/2019 strain isolate. The deduced amino acids sequences' comparative alignment of the five Avian OrthoAvulavirus-1 isolates confirmed the presence of motif of virulent strains (RRQKRF) between 112 and 117 residues of fusion protein cleavage site. All five AOAV-1 isolates from 2019 and 2020 belonged to the NDV genotype classified as genotype VII.1.1 depending on full Fusion gene sequences; those five isolates were grouped with subgenotype VIId. Through significant genetic changes in vNDV observed in our study, continuous monitoring of the multiple vNDV outbreaks in vaccinated chicken and targeted evaluation of the used vaccines.

Heterologous prime-boost vaccination programs against Newcastle disease virus genotype VII in chickens

The effectiveness of two vectored ND vaccines (Innovax® ND and Vectormune® ND) was tested, in single and prime-boost vaccination programs, against challenge with velogenic NDV genotype VII, in eight chicken groups (G). G1 and G2 were control groups. G3–5 received Innovax® ND, while G6–8 received Vectormune® ND on the hatchery. Additionally, G4 and G7 received live La Sota two-weeks later, and G5 and G8 received inactivated vaccine one-week later. On the challenge day, G3 (Innovax® ND vaccine alone) had lower HI titer than G6 (Vectormune® ND vaccine alone) (p < 0.05). Boosting with inactivated vaccine instead of La Sota increased serological response. G3 had the same HI titer as G2 (positive control) but decreased viral shedding significantly after the 3rd day post challenge (d pc). G8 (Vectormune® ND plus inactivated vaccine) had 86.5 % decrease of viral shedding on the 3rd d pc, while that of G7 (Vectormune® ND plus La Sota) persisted until the 6th d pc. All regimens increased weight gain (p < 0.05), fully protected against mortality, and showed few clinical signs. Prime-boost vaccines provided significant reduction and time shortening of heterologous viral shedding.

Protection against genotype VII Newcastle disease virus challenge by a minicircle DNA vaccine coexpressing F protein and chicken IL-18 adjuvant

Genotype VII Newcastle disease virus (NDV) is still one of the most important virus threats severely affecting poultry production worldwide. Although inactivated vaccines are commercially available, there is still an urgent need to develop novel vaccine candidates for convenient and affordable vaccine application. Oral immunization using live attenuated bacteria such as Salmonella has recently attracted increasing interest, and in a previous study, we used a regulated delayed lysis Salmonella vector to deliver a DNA vaccine encoding the F protein and chicken IL-18 adjuvant together, named pYL23. To further improve its efficiency, we employed a novel in vivo minicircle DNA (mcDNA) platform to construct pYL58, which could maintain the complete plasmid during in vitro culture conditions and then transform into mcDNA in vivo whenever the plasmid was delivered by Salmonella into host cells. Compared with immunization with the parental strain harboring plasmid pYL23, immunization with Salmonella with pYL58 induced increased levels of serum IgY and mucosal sIgA in chickens, especially the intestinal and tracheal sIgA levels. Production of cytokines, including IL-4, IFN-γ, IL-18 and IFN-α, was also determined in serum and spleen cell culture supernatants after the 3rd immunization, and the results showed that the production of IFN-γ in the pYL58 group was significantly increased compared with that in the negative control group. Interestingly, compared with pYL23, significantly increased production of IFN-α in the cell supernatants from the pYL58 group was also observed. In addition, the CCK-8 assay results showed that the minicircle pYL58 significantly increased spleen cell proliferation. After virulent VII NDV challenge, pYL58 immunization could provide 70% protection compared with 50% protection in the pYL23 group, together with decreased virus titers in chicken lung samples at Day 5 and virus shedding at Days 3 and 5 post-challenge. This study demonstrated that the application of mcDNA technology dramatically increased the DNA vaccine efficiency, providing additional support for the use of our mcDNA platform in the veterinary field.

Construction and immune efficacy of a recombinant turkey herpesvirus vaccine strain expressing fusion protein of genotype VII Newcastle disease virus

Herpesvirus of turkeys (HVT), a commonly used live vaccine against Marek’s disease, has proven to be a highly effective viral vector for the generation of recombinant vaccines that deliver protective antigens of other avian pathogens. In this study, a vaccine designated rHVT-NDV-opti F was constructed by inserting a codon-optimized genotype Ⅶ Newcastle disease virus (NDV) fusion (F) gene into the US2 gene of HVT Fc126 vaccine strain using CRISPR/Cas9 gene-editing technology coupled with two single-guide RNAs (sgRNA). The F protein expression of rHVT-NDV-opti F was detectable by western blotting and an indirect immunofluorescence assay. Compared with wildtype HVT, rHVT-NDV-opti F has similar plaque morphology but lower in vitro replication capacity. The F protein of rHVT-NDV-opti F is genetically stable and predominantly expressed in the cell plasma. Immunization of one-day-old specific pathogen-free chickens with 4000 plaque-forming units of rHVT-NDV-opti F induced NDV-specific antibodies and provided 70% protection against a homologous NDV challenge, effectively reducing virus shedding, clinical signs, tissue viral load, and mortality. These results suggest that rHVT-NDV-opti F could be a potential vaccine candidate against Newcastle disease in chickens and that HDR-CRISPR/Cas9 combined with dual sgRNA can rapidly and efficiently construct HVT-vectored vaccine candidates.

Alteration in the Population of Intraepithelial Lymphocytes and Virus Shedding in Specific-Pathogen-Free Chickens Following Inoculation with Lentogenic and Velogenic Newcastle Disease Virus Strains.

Intraepithelial lymphocytes (IELs) provide the first line of immunological defense after the invasion of the intestine by a pathogen. To understand the changes of IEL response in chickens, we measured the population of different subsets of avian IELs at different time points after primary inoculation of Newcastle disease virus (NDV) lentogenic strain (LaSota) and subsequent challenge with NDV velogenic strain- genotypes VII and VIII. Furthermore, NDV shed after each treatment was quantified. Specific-pathogen-free chickens were randomly divided into six groups of chickens, one to six, inoculated with phosphate buffered saline; NDV lentogenic strain (LaSota); genotype VII (GVII); LaSota and challenged with GVII (LSGVII); genotype VIII (GVIII); and group of LaSota and challenged with GVIII (LSGVIII). The chickens were euthanized at 12, 36, and 60 h postchallenge. Immunophenotyping of CD25+ IEL, CD3+ cells, CD4+ cells, and CD8+ cells was conducted using flow cytometer. Furthermore, virus shedding was measured using reverse transcriptase-quantitative polymerase chain reaction. Data were analyzed using a two-way analysis of variance (ANOVA). The results showed that the percentage population of IEL subsets was generally lower in the chickens inoculated with GVII or GVIII when compared with LaSota, LSGVII and LSGVIII inoculated groups. The NDV copy number was significantly higher in chickens challenged with NDV GVII or GVIII when compared with chickens inoculated with LaSota, LSGVII or LSGVIII. Taking together, NDV velogenic strain caused decrease in the population of subsets of chickens' IEL. However, inoculation of NDV LaSota may increase the population of avian IEL subsets and decrease shedding of virulent NDV.

Newcastle disease virus genotype VII gene expression in experimentally infected birds

Newcastle disease virus genotype VII (NDV-GVII) is a highly contagious pathogen responsible for pandemics that have caused devastating economic losses in the poultry industry. Several features in the transcription of NDV mRNA, including differentially expressed genes across the viral genome, are shared with that for other single, non-segmented, negative-strand viruses. Previous studies measuring viral gene expression using northern blotting indicated that the NDV transcription produced non-equimolar levels of viral mRNAs. However, deep high-throughput sequencing of virus-infected tissues can provide a better insight into the patterns of viral transcription. In this report, the transcription pattern of virulent NDV-GVII was analysed using RNA-seq and qRT-PCR. This study revealed the transcriptional profiling of these highly pathogenic NDV-GVII genes: NP:P:M:F:HN:L, in which there was a slight attenuation at the NP:P and HN:L gene boundaries. Our result also provides a fully comprehensive qPCR protocol for measuring viral transcript abundance that may be more convenient for laboratories where accessing RNA-seq is not feasible.

Single-Dose Vaccination of Recombinant Chimeric Newcastle Disease Virus (NDV) LaSota Vaccine Strain Expressing Infectious Bursal Disease Virus (IBDV) VP2 Gene Provides Full Protection against Genotype VII NDV and IBDV Challenge.

Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV) are the two most important and widespread viruses causing huge economic losses in the global poultry industry. Outbreaks of genotype VII NDV and IBDV variants in vaccinated poultry flocks call for genetically matched vaccines. In the present study, a genetic matched chimeric NDV LaSota vaccine strain expressing VP2 gene of IBDV variant, rLaS-VIIF/HN-VP2 was generated for the first time, in which both the F and HN genes of LaSota were replaced with those of the genotype VII NDV strain FJSW. The cleavage site of the FJSW strain F protein in the rLaS-VIIF/HN-VP2 genome was mutated to the avirulent motif found in LaSota. Expression of IBDV VP2 protein was confirmed by western blot. The rLaS-VIIF/HN-VP2 maintained the efficient replication ability in embryonated eggs, low pathogenicity and genetic stability comparable to that of parental LaSota virus. One dose oculonasal vaccination of one-week-old SPF chickens with rLaS-VIIF/HN-VP2 induced full protection against genotype VII NDV and IBDV lethal challenge. These results indicate that the rLaS-VIIF/HN-VP2 is a promising bivalent vaccine to prevent infections of IBDV and genotype VII NDV.

Comparison of the protective antigen variabilities of prevalent Newcastle disease viruses in response to homologous/heterologous genotype vaccines

The genotype VII Newcastle disease virus (NDV) vaccine has begun to replace the traditional genotype II NDV vaccine and is widely used in the commercial poultry of China. However, the effect of homologous and heterogeneous anti-NDV serum on the evolution of prevalent NDV is unknown. To understand the effect of genotype II and VII anti-NDV serum on the evolution of genotype VII NDV strains, ZJ1 (waterfowl origin) and CH/SD/2008/128 (ND128; chicken origin) were used for serial passage of 30 generations in DF-1 cells without anti-NDV serum or with genotype II and VII anti-NDV serum independently. The F and HN genes of the 2 viruses were amplified for the 10th, 20th, and 30th generations of each serial passage group and compared with their respective original viruses. We found that there was only one mutation at position 248 in the F gene of ZJ1 due to the serum pressure of genotype VII anti-NDV. Similarly, mutations at residue 527 of the F gene, and position 9 and 319 of the HN gene of ND128 were noted in both anti-NDV serum groups. The results show that the nonsynonymous (NS)-to-synonymous (S) ratio of the F gene of ZJ1 virus was 1.6, and for the HN gene, it was 2.5 in the anti-II serum group. In the anti-VII serum group, the NS/S ratio for the F gene was 2.1, and for the HN gene, it was 2.5. The NS/S ratio of the F gene of the ND128 virus was 0.8, and for the HN gene, it was 3 in the anti-II serum group. Furthermore, the NS/S ratio of the F gene was 0.8, and the HN gene was 2.3 in the anti-VII group. Taken together, our findings highlight that there was no significant difference in the variation of protective antigens in genotype VII NDV under the selection pressure of homologous and heterogeneous genotype NDV inactivated vaccines.