Identification of a potential neutralizing linear epitope of hemagglutinin-neuraminidase in Newcastle disease virus.

BACKGROUND: The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a major antigen that can induce protective antibodies in poultry. However, its antigenic epitopes have not been fully elucidated. Therefore, defining the linear epitopes of HN, especially neutralizing epitopes, will be useful for revealing its antigenic characterization. METHODS: In this study, we analyzed B-cell immunodominant epitopes (IDEs) of the HN protein from the vaccine strain LaSota using pepscan technology with LaSota-specific chicken hyperimmune antisera. We constructed IDEs-RFP plasmids and prepared anti-IDEs peptide mouse sera to identify IDEs through immunological tests. At last, the different diluted anti-IDE antisera were used in BHK-21 cells to perform the neutralization test. RESULTS: Five IDEs of the HN were screened and further verified by indirect immunofluorescence assays, dot blots and Western blots with NDV- and IDEs-specific antisera. All five IDEs showed good immunogenicity. IDE5 (328-342 aa) could recognize only class II NDV but did not react with the class I strain. Most of the IDEs are highly conserved among the different strains. A neutralization test in vitro showed that the peptide-specific mouse antisera of IDE4 (242-256 aa) and HN341-355, a reported neutralizing linear epitope, could partially neutralize avirulent LaSota as well as virulent strains at similar levels, suggesting that IDE4 might be a potential neutralizing linear epitope. CONCLUSION: The HN protein is a major protective antigen of NDV that can induce neutralizing antibodies in animals. We identified five IDEs of the HN using a pepscan approach with NDV-specific chicken hyperimmune antisera. The five IDEs could elicit specific antibodies in mice. IDE4 (242-256 aa) was identified as a novel potential neutralizing linear epitope. These results will help elucidate the antigenic epitopes of the HN and facilitate the development of NDV vaccines.

Identification of Two Distinct Linear B Cell Epitopes of the Matrix Protein of the Newcastle Disease Virus Vaccine Strain LaSota.

Matrix (M) protein of Newcastle disease virus (NDV) is an abundant protein that can induce a robust humoral immune response. However, its antigenic epitopes remain unknown. In this study, we used a pepscan approach to map linear B cell immunodominant epitopes (IDEs) of M protein with NDV-specific chicken antisera. The six epitopes with the highest reactivity by peptide scanning were obtained as IDE candidates. Among them, aa71-85 and aa349-363 were identified by immunological assays with NDV-specific or IDE-specific antisera. The minimal antigenic epitopes of the two IDEs were further characterized as 77MIDDKP82 and 354HTLAKYNPFK363. Moreover, an amino acid sequence alignment and immunoblot analysis revealed the conservation of the two IDEs in the M protein of strains of different genotypes. These two IDEs of M protein could be genetically eliminated as negative markers in recombinant NDV for serologically differential diagnosis in the development of marker vaccines.

The intracorporal interaction of fowl adenovirus type 4 and LaSota strain significantly aggravates the pathogenicity of one another after using contaminated Newcastle disease virus-attenuated vaccine.

In the past five years, inclusion body hepatitis-hydropericardium syndrome caused by fowl adenovirus type 4 (FAdV-4) has been rampant in China and is still an epidemic at present. We had found the contamination of FAdV-4 in Newcastle disease virus (NDV)-attenuated vaccine from a large farm with aforementioned disease, and then conjectured that the use of this contaminated vaccine might be an important reason for the outbreak of the FAdV-4 infection. To assess the pathogenicity of this contaminated vaccine and the interaction between FAdV and LaSota, systemic animal experiments were launched with FAdV infection via the contaminated vaccine, as well as FAdV direct infection. Results showed that no chicks died after FAdV direct infection, while a 16.7% mortality rate appeared after FAdV infection through the contaminated vaccine; the synergistic reaction of FAdV and LaSota aggravated their inhibitory action for weight growth of SPF chickens, enhanced one another's capacity for immune system destruction, promoted their multiplication in vivo, and restrained antibody production. This study demonstrated the intracorporal interaction and enhanced pathogenicity of FAdV-4 and LaSota strain in contaminated NDV-attenuated vaccine, illustrating how the exogenous virus causes infection and induces severe diseases, which reminds us that the damage of attenuated vaccine contaminated with FAdV-4 even in extremely low dose is not insignificant.

Validation of real-time reverse transcription polymerase chain reaction to detect virus titer and thermostability of Newcastle disease live virus vaccine.

BACKGROUND AND AIM: Newcastle disease is one of the most common diseases affecting poultry in Bangladesh. The disease can cause up to 100% mortality but is preventable if birds are timely and properly vaccinated with a vaccine of standard virus titer. Different live vaccines are commercially available in the country - most, if not all, are produced using lentogenic strains of the virus with variable virulence. One of the disadvantages of these vaccines is that they are not stable at high environmental temperature, and therefore, a proper cold chain must be maintained during transportation and storage. Information on how long these vaccine viruses can withstand environmental temperature, which is near the vicinity of 37°C in the summer season in Bangladesh, is scanty. The aim of this research was to measure the effect of temperature on virus titer of live ND virus vaccines and to develop a real-time reverse transcription polymerase chain reaction (rRT-PCR) standard curve to indirectly determine hemagglutination (HA) titer of virus by this highly sensitive method. MATERIALS AND METHODS: In this study, thermostability of five commercial live vaccines containing LaSota, F, Clone 30, and B1 type LaSota strains was observed for up to 35 days keeping them at 37°C. From the most thermostability yielding sample, two rRT-PCR standard curves were developed: (1) By plotting the cycle threshold (CT) values as obtained from 10-fold serial dilutions up to 10-3 against their corresponding log (to the base 10) dilutions and (2) by plotting the CT values obtained from serial HA dilutions up to 2-4 against their corresponding HA titer dilutions. The PCR efficiencies based on which the graphs were fitted were also evaluated. RESULTS: The vaccine from the LaSota strain withstood 37°C for 35 days with a gradual declination of HA titer over time, and this vaccine also had the highest initial HA titer, which was 211. The vaccine made from F strain was inactivated quickly, and it had the lowest HA titer at the beginning of the study. The first standard curve developed can be used to assess the level of virus titer in a diluted sample compared with the titer in the original undiluted vaccine preparation by plotting the CT value obtained from the dilution by rRT-PCR. The second standard curve can be used to calculate the HA titer of a vaccine dilution by plotting the CT value as obtained from the dilution by rRT-PCR. CONCLUSION: The regression equations for the first and second graphs were y=-3.535x+14.365 and y=-1.081x+13.703, respectively, suggesting that, for every 3.53 cycles, the PCR product would have increased 10 times and 2 times for every 1.08 cycles, respectively, indicating nearly (but not exactly) 100% PCR efficiency.

Vaccination of broilers against Newcastle disease in the presence of maternally derived antibodies.

OBJECTIVE: The aim of this study was to evaluate the efficacy of a live attenuated vaccine against Newcastle disease in broilers with different levels of maternally derived antibodies (MDA). While vaccination remains the single most important means for controlling Newcastle disease, presence of MDA may interfere with the vaccination of young birds and decrease the efficacy of the vaccine. MATERIALS AND METHODS: Day-old chicks with variable levels of MDA (negative, low and high) were vaccinated with a live attenuated vaccine against Newcastle disease. Three most commonly used inoculation routes were compared; oculonasal, spray and oral (drinking water). Onset and duration of immunity were measured by serology and challenge with virulent virus. RESULTS: Immune response in vaccinated MDA-positive birds was delayed in comparison with SPF controls. Protection was well established already at 14 days post vaccination in SPF birds while in MDA-positive birds it was 1-2 weeks delayed and was lower throughout the study. Non-vaccinated MDA-positive birds lost passive protection completely at 3-4 weeks of age and were significantly more susceptible to challenge than vaccinated hatch mates at all test points. The protection rate increased in vaccinated birds towards the end of the experiment and reached 70-100 % at the last test points (35-42 days of age). Correlation of haemagglutination inhibition (HI) titre vs. protection rate revealed the importance of cellular and local immunity as most of the vaccinated birds with low HI titre were protected, contrary to their unvaccinated hatch mates with the same HI titre. Oculonasal route seems to provide slightly better protection than the other two routes. CONCLUSIONS AND CLINICAL RELEVANCE: Although immune protection in vaccinated MDA-positive birds may be decreased or delayed, vaccination still provides high protection against ND challenge in comparison with the unvaccinated hatch mates. The degree of interference seems to be proportional to the level of MDA. Vaccination schedules therefore need to be designed according to the immune status of the flock.

Recombinant Newcastle disease virus expressing the infectious bronchitis virus S1 gene protects chickens against Newcastle disease virus and infectious bronchitis virus challenge.

The recombinant LaSota strain expressing a chimeric IBV S1 gene (rLaSota-S1) was constructed with the S1 gene of the LX4 type IBV ck/CH/LDL/091022. The expression of the S1 protein was detected by an indirect immunofluorescence assay and Western blotting. The rLaSota-S1 strain was slightly attenuated, and its growth dynamics were similar to that of the parental LaSota strain. Vaccination of specific pathogen-free chickens with the rLaSota-S1 strain induced NDV hemagglutination inhibition antibodies, and it protected chickens from challenge with virulent NDV. In addition, vaccination with the rLaSota-S1 strain induced IBV-specific IgG antibodies and cellular immunity; however, a single vaccination provided partial protection with reduced virus shedding. Better protection efficiency was observed after a booster vaccination, which resulted in higher antibody titers, significantly fewer disease symptoms, and reduced virus replication and shedding. Our results suggest that the rLaSota-S1 strain is a bivalent vaccine candidate against both NDV and IBV.