Improved immunogenicity of Newcastle disease virus inactivated vaccine following DNA vaccination using Newcastle disease virus hemagglutinin-neuraminidase and fusion protein genes

The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.     

表达新城疫HN和F基因重组禽痘病毒的免疫效力测定

将8日龄SPF鸡随机分组.分别接种rFPVHN、rFPVF、rFPVNHF重组禽痘病毒,接种量为每只鸡105PFU,另一组群用La Sota活苗经点眼、滴鼻接种10<'6>EID<,50>.于免疫后6d、12d和18d测定HI和微量中和抗体.结果发现,3种重组病毒均能刺激鸡群产生抗新城疫病毒特异抗体,但抗体产生时间比La Sota活苗缓慢,且抗体滴度亦低.然而共表达HN和F的rFPVNHF免疫鸡群的中和抗体水平明显比rFPVHN和rFPVF组群高.表明rFPVNHF的保护性免疫更优于HN或F单基因重组禽痘病毒.     

Antibody reaction to inhalation vaccination of poultry against Newcastle disease]

Aerosol preparated from the AVIPEST vaccine by means of three types of aerosol generators was used for vaccination of 294 chickens of different age against the Newcastle disease in laboratory tests and 30 000 chickens at the age of two and three weeks under field conditions. The general and local antibody reactions to vaccination were evaluated on the basis of the determination of the content of hemagglutination-inhibition and neutralization antibodies in the blood serum and in respiratory-tract flushings. The level of secretory antibodies in the flushings from the respiratory tract, determined by the described original method, is an important parameter of the resistance level of the organism to infection. The highest average levels of antibody titres in serum and in flushings were obtained 14 to 21 days from vaccination (7-10 log2). The La Sota vaccination strain was eliminated from the organism after spray vaccination and immunized the non-vaccinated contact chicken. Revaccination increased and greatly prolonged immunity. All the chickens inoculated showed resistance to challenge infection whereas the control birds died within four to six days.     

新城疫病毒HN蛋白抗原表位分析及结构域基因原核表达

以本实验室构建的含新城疫病毒血凝素-神经氨酸酶基因重组质粒为模板，设计引物通过PCR和重叠-延伸PCR扩增，获得HNa、HNb和HNa-L-b三种抗原结构域基因片段，BamHⅠ／HindⅢ双酶切定向克隆到原核表达载体pET32c，获得重组质粒分别命名为pET32c—HNa、pET32c-HNb和pET32c．HNa—L—b。重组质粒转化大肠杆菌感受态细胞BL21，筛选出阳性克隆，诱导表达并取产物进行分析。结果表明，HNa、HNb、HNa-L-b结构域基因片段均获得了融合表达。Western-blotting分析证实表达产物HNa和HNb与NDV阳性血清具有免疫反应性。本试验结果为进一步研究HN蛋白抗原结构域的免疫原性以及HN蛋白与F蛋白在细胞融合中的相互作用奠定了基础。     

新城疫病毒F蛋白抗原表位串联基因的构建及其原核表达

用自行设计的3对引物,通过RT-PCR从接毒的SPF鸡胚的尿囊液中克隆了新城疫病毒F基因3段抗原表位区段,大小分别为81、108、105bp。应用添加互补性酶切位点的基因工程方法,将3段抗原表位基因片段拼接成多表位串联基因,大小为306bp。将此基因片段插入含组氨酸(His)基因的质粒pET-32-a中,构建了重组质粒pET-32-a-F306。经诱导表达,获得相对分子质量约为31000的融合蛋白,其中F蛋白抗原表位串联基因片段表达产物约为11000。经亲和层析,获得纯化His-F306融合蛋白,进一步用该蛋白免疫小鼠,制备了鼠源抗新城疫病毒F蛋白抗体。经过琼扩、ELISA及Western-blot检测,表明该融合蛋白中的抗原表位串联基因所表达的蛋白具有良好的抗原性。     

Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2¹³ per 25 µL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4℃. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.     

以rLaSota为骨架构建表达强毒株HN蛋白的嵌合体新城疫病毒

新城疫病毒(NDV)的HN蛋白是一个多功能蛋白,具有血凝素(HA)和神经氨酸酶(NA)两种活性,在病毒感染过程中扮演重要角色。本研究利用反向遗传操作技术,将NDV强毒株F48E9的HN基因替换弱毒株rLaSota的HN基因,获得嵌合病毒rL-F48E9HN。收获病毒尿囊液,提取基因组RNA,进行序列分析,结果显示嵌合病毒基因组的HN基因获得了正确替换。嵌合病毒在鸡胚内的生长特性与亲本株LaSota一致,与F48E9的生长特性相差甚远。嵌合病毒红细胞吸附性明显增强,较rLaSota株增加51%,而细胞融合作用无明显变化。rL-F48E9HN的鸡胚平均致死时间(MDT)为148 h,脑内致病指数(ICPI)为0.43,静脉接种致病指数(IVPI)为0,说明rL-F48E9HN的毒力仍然属于弱毒力范围,而没有达到中等毒力或者强毒力。     

鹅源新城疫病毒JG97株HN基因的克隆与遗传分析

根据GenBank中公开的鸡新城疫病毒(NDV)血凝素-神经氨酸酶(HN)基因序列合成了1对引物,通过RT-PCR扩增出了鹅源NDV JG97分离株的HN基因.将扩增产物克隆到pGEM-T载体中,并进行了序列测定.结果表明,扩增的基因片段含1个完整的、长1 716 bp的HN基因阅读框(ORF),编码的571个氨基酸中含13个Cys残基和5个潜在的糖基化位点.同源性分析结果表明,JG97株与SF02、GD/1/98/Go、JS/1/97/Go、JS/2/98/Go、JS/5/01/Go、NA-1、SD/5/04/Go和ZJ/1/00/Go等8个毒株的HN基因和氨基酸同源性分别为96.3%～98.2%和97.0%～98.9%,而与F48E9株分别为84.8%和89.8%,与La Sota株分别为82.1%和86.9%,表明JG97株与我国其他鹅源NDV毒株的亲缘关系较近,它们可能具有共同的来源,但与经典NDV毒株间差异较大.此外,鹅源NDV毒株HN蛋白中有6个独特的氨基酸变异,另有14个仅与Taiwan95和VOL95毒株共有的氨基酸变异.     

Effect of tuftsin on embryo vaccination with Newcastle disease virus vaccine

The effect of tuftsin of embryo and post-hatch vaccination with NDV-F was studied. The embryo vaccination with NDV-F resulted in more number of dead-in-shell embryos. To overcome this problem, the vaccine was treated separately with ethyl methane sulfate (EMS) and 5-fluorouracil (5-FU) and administered. Treating the vaccine with 5-FU resulted in better hatchability as compared to EMS treatment. In embryo, NDV antibody titres increased upto 2 weeks of age and declined thereafter, whereas in post-hatch vaccination, the antibody titre increased from second to fourth week of age and declined thereafter. The seroconversion was better when the vaccine was given along with tuftsin either to embryos or chicks (post-hatch vaccination) as compared to those vaccinated without tuftsin. Moreover, the percentage of hatchability was more in tuftsin administered groups. It was found that embryo vaccination can ensure definite protection during the early life of the chicks despite the presence of maternal antibodies. In cases where breeder vaccinations do not result in concomitant transfer of antibody to progeny chicks, embryo vaccination would give only neonatal resistance. During the later stages, embryo vaccination did not confer any advantage over post-hatch vaccination.     

Serological response of chickens to oral vaccination with Newcastle disease virus

Conventional Newcastle disease vaccines are not suitable for application to village chickens in tropical countries of Asia. Trials with food-based vaccines are being initiated and the following experiments were performed to evaluate oral vaccination with Newcastle disease virus. Experimental chickens were vaccinated orally with the avirulent V4 strain of Newcastle disease virus and haemagglutination-inhibition antibody responses were measured. V4 virus was introduced into the crop by tube and total faecal output was collected daily and assayed for Newcastle disease virus. Virus was recovered on Days 5 and 6 after vaccination from most chickens that had received 10(7.4) and 10(6.4) 50% egg-infectious doses (EID50) of virus. There was no recovery of virus from birds receiving a lower dose of vaccine. Groups of chickens kept in cages with wire floors were given various doses of vaccine into the crop. Higher antibody titres were achieved with higher doses of virus. This dose responsiveness was not observed when various doses of vaccine were presented on food pellets and the groups of chickens were kept on concrete floors. Similar antibody responses were then seen with nominal doses of 10(5.2) and 10(8.2) EID50 per bird, possibly as a result of excretion and re-ingestion of the vaccine virus. Spread of the vaccine virus was demonstrated when control chickens and chickens receiving 10(7.7) EID50 of V4 virus on food pellets were housed together on a concrete floor. Similar antibody titres were achieved in both vaccinated and in-contact chickens.     

Protection conferred by vaccination with Blacksburg and Komarov strains of Newcastle disease virus against Newcastle disease in Bangladesh

Summary An evaluation was undertaken of the efficacy of vaccination of day-old chicks with the Blacksburg (B1) strain of Newcastle disease virus (NDV) followed at various times by vaccination with the Komarov (K) strain. Antibody was detected by the haemagglutination inhibition (HAI) test one week after vaccination with B1 and titres peaked at three weeks and had declined to undetectable levels by nine weeks.After subsequent vaccination with K strain at five, seven or eight weeks of age levels of HAI antibody (titre 80 to 640) were detected after three weeks. Birds vaccinated at seven weeks were tested for antibody and resistance to challenge beyond 19 weeks of age. In this group the HAI titres remained constant (80 to 640) up to 32 weeks of age and then steadily declined to 10 to 20 at 44 weeks of age.A linear relationship between HAI titre and virus neutralising index (VNI) was demonstrated with a range of selected sera. Only birds with an HAI titre of 80 or greater resisted artificial challenge. It is recommended that, following B1 vaccincation at day-old and K vaccination at seven weeks old, revaccination with K strain should be performed at intervals of not more than seven months.

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Proteccion Conferida Mediante Vacunacion Con Las Cepas Blacksburg Komarov Del Virus De Newcastle En Bangladesh

Resumen Se llevó a cabo una evaluación de la eficacia de la vacunación de pollitos de un día, con la cepa Blacksburg (B1) del virus de la enfermedad de Newcastle, seguida de varias aplicaciones vacunales periódicas con la cepa Komarov (K). Se detectaron anticuerpos mediante la prueba de inhibición de la hemaglutinación, una semana después de la vacunación con B1, los títulos alcanzando el pico a las tres semanas y declinaron a niveles no detectables a las nueve semanas.Después de aplicaciones vacunales periódicas con la cepa K, a las cinco, siete u ocho semanas de edad, se detectaron anticuerpos mediante la prueba de inhibición de la hemaglutinación (títulos 80 a 640), después de tres semanas. Las aves vacunadas a las siete semanas, recibieron una descarga y se les hizo prueba de anticuerpos, después de las 19 semanas de edad. En este grupo los títulos de anticuerpos permanecieron constantes (80 to 640) hasta las 32 semanas de edad y después declinaron lentamente a 10 ó 20 a las 44 semanas de edad.Se demostró una relación lineal entre los títulos hallados y el índice viral neutralizante, en un rango de sueros seleccionados. Solamente las aves con títulos de 80 o más altos resistieron la descarga artificial. Se recomienda que, seguidamente a la vacunación con B1 a un día de edad y con K a las siete semanas, las revacunaciones con la cepa K debería hacerse con intervalos de no más de siete meses.

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Protection Conferee Contre La Maladie De Newcastle Au Bangladesh Par La Vaccination A L'Aide Des Souches Blacksburg Et Komarov De Virus De La Maladie De Newcastle

Résumé Une evaluation de l'efficacité de la vaccination de poussins d'un jour à l'aide des souches Blackburg (B1) du virus de la maladie de Newcastle, suivie à différents moments par la vaccination à l'aide de la souche Komarov (K), a été entreprise. Les anticorps furent détéctés par inhibition de l'hemagglutination (HAI), une semaine après la vaccination avec la souche B1 et le titre atteignit un pic à 3 semaines pour décliner jusqu'à des niveaux indétectables à neuf semaines. A la suite de vaccinations consécutives avec la souche K à cinq, sept ou huit semaines, les niveaux d'anticorps HAI furent détectés après trois semaines. Les oiseaux vaccinés à sept semaines furent examinés pour les anticorps et la résistance à une épreuve au delà de 19 semaines d'âge. Dans ce groupe les titres HAI sont restés constants (80 à 640) jusqu'à l'àge 32 semaines, puis ont diminué régulièrement jusqu'à 10 à 20 à l'âge de 44 semaines.Une relation linéaire entre le titre HAI et l'index de neutralisation du virus (VNI) a été démontrée sur une gamme de sérums choisis. Seuls les oiseaux ayant un titre HAI de 80 ou plus ont résisté à l'épreuve artificielle. On recommande que, après une vaccination B1 à un jour, et une vaccination K à l'âge de sept semaines, une revaccination K soit réalisée à des intervalles n'excédant pas sept mois.