Isolation and biological properties of virulent subpopulations from lentogenic Newcastle disease virus strains

From each of two lentogenic Newcastle disease virus (NDV) strains of the type LaSota and Hitchner B1 a virulent subpopulation could be obtained. The two subpopulations were—in comparison to the two parent viruses—more resistant to the lipid solvent chloroform and more stable against thermal degradation. Also, the glycoproteins haemagglutinin and F (fusion) were more stable against thermal inactivation. Electron microscopic observations revealed in terms of size and morphology all of the characteristics of NDV. Both subpopulations possessed, however, the same elution kinetics as their respective parent strains. The intracerebral and intravenous pathogenicity indices as well as the mean death times of the two subpopulations allow to classify these viruses as virulent Newcastle disease viruses.     

Comparison of the immunogenicity of Newcastle disease virus strains V4, Hitchner Bl and La Sota in chickens

The immunogenicity of three Newcastle disease virus (NDV) strains--V4, Hitchner B1 and La Sota, was assessed in chickens that had varying levels of maternal antibody to the virus. Chickens were immunised at different ages by the eye drop and aerosol methods. The immune response of chickens to similar doses of the strains was affected by the presence of maternal antibody. Both the level of maternal antibody and the strain of virus used affected the result. Strain La Sota was least affected as it took higher levels of maternal antibody to depress response to it than were required to have a similar effect on Hitchner B1 and V4. Strain V4 was the strain most affected by circulating maternal antibody. The results demonstrated that strain V4 was less immunogenic than Hitchner B1 and La Sota when used in chickens with maternal antibody to NDV.     

Antibody response to strain combinations of Newcastle disease virus as measured by hemagglutination-inhibition

Differences in antibody response to three Newcastle disease virus (NDV) strains--B-1, LaSota, and Ulster--were investigated using the hemagglutination-inhibition (HI) micro-titer test in chickens hatched from ND-immune and unimmune flocks. When used singly as primary vaccines, the Ulster strain stimulated the lowest antibody response of the three in both immune and unimmune (susceptible) chickens. Subgroups of each of the primary-vaccinated groups were revaccinated with each of the three strains. Ulster-vaccinated chicks, revaccinated with Ulster, gave the poorest booster response. All other revaccination combinations gave a significant titer increase, though some were better than others. It is suggested that the Ulster strain as primary vaccine followed by booster does of B-1 or LaSota will induce a higher antibody response (i.e., immunity) in susceptible chicken populations with less risk of a post-vaccination reaction.     

新城疫病毒西藏分离株的生物学特性鉴定及遗传进化分析

从西藏病死藏鸡中分离到具有血凝活性的病毒、经血凝抑制试验、电镜观察、PCR扩增和测序鉴定为新城疫病病毒(NDV),通过动物致病性试验证明该病毒对鸡具有致病性;分离毒株毒力测定结果显示,MDT为120h,EID50为10-8.44、IVPI为0.5、ICPI为0.6,均符合NDV弱毒株特征。血凝解脱及血凝素热稳定性试验显示:各分离株的血凝解脱时间短,血凝素热稳定性较差,符合NDV弱毒株的特征。F基因的序列测定遗传进化分析表明,西藏分离毒株之间的核苷酸序列具有99%的同源性,与疫苗株LaSota的同源性为90%;与国内标准强毒株F48E8同源性为81%。推导其氨基酸序列分析表明,各分离株的F蛋白的裂解位点氨基酸112 G-K-Q-G-R-L117,具有NDV弱毒株特征,与毒力测定结果相符。本研究首次报道了NDV西藏分离毒株遗传进化情况和生物学特性情况,为进一步研究高海拔、缺氧环境下NDV生物学特性变化研究奠定了基础。     

鸡新城疫、传染性支气管炎、禽流感(H9亚型)三联灭活疫苗对禽流感H9亚型流行株攻毒的保护作用

为了监测鸡新城疫、传染性支气管炎、禽流感(H9亚型)三联灭活疫苗(LaSota株+M41株+SS/94株)对H9亚型禽流感病毒流行毒株的免疫保护效果,采用H9亚型禽流感病毒SS/94株及2009—2010年现地分离的3株H9亚型禽流感病毒对已免疫上述三联灭活苗的SPF鸡进行攻毒试验。结果显示,试验鸡以0.3 mL/只的剂量免疫三联灭活苗后21 d,其H9亚型禽流感病毒的HI抗体效价可达8～11log2,此抗体水平可抵抗2×106EID50的H9亚型禽流感病毒SS/94株、BLCN09株、WDZ09株、YT10株的攻击,攻毒保护率均达90%(9/10)以上。可见,以SS/94株作为禽流感疫苗抗原制备的三联灭活苗具有良好的免疫原性,能使免疫鸡抵抗2009—2010年期间现地分离的多株H9亚型禽流感病毒的攻击。     

Immunogenicity and protective efficacy of virosome based vaccines against Newcastle disease

Virosome based vaccines against Newcastle disease (ND) were prepared and evaluated for their immunogenicity and protective efficacy in chickens. Envelop of Newcastle disease virus (NDV) was solubilised with Triton X-100 to yield virosomes which were later on encapsulated in poly-lactide-co-glycolide (PLG) microspheres. The birds were immunized intranasally with virosomes or PLG microspheres encapsulated virosomes, and efficacy of these preparations was compared with commercial LaSota vaccine. The preparations protected the chickens against virulent virus challenge infection, however the microencapsulated virosome vaccine gave slightly lesser degree of protection than non encapsulated counterpart. The humoral and cell mediated immune response generated as well as the protection afforded by virosome preparations were found to be comparable with LaSota vaccine. The results substantiate the potential of virosome based vaccines to provide high level of immunity and protection against Newcastle disease.     

新城疫病毒单克隆抗体的制备及与不同分离株的反应性

以纯化的新城疫病毒(Newcastle dis-ease virus,NDV)弱毒株La Sota为免疫原,接种6周龄~8周龄Balb/c小鼠。最后一次加强免疫后3 d,取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合。以纯化的NDV建立了筛选单抗的间接ELISA,经多次检测,3次亚克隆,共获得5株NDV特异性单抗,这5株单抗与NDV不同分离株的反应性存在差异,表明部分NDV毒株抗原性发生了变异。     

A controlled vaccination trial for Newcastle disease under village conditions

All the poultry in each of four distinct Moroccan villages were vaccinated against Newcastle disease using Hitchner B1 and inactivated vaccines. Poultry in a fifth village were monitored as controls. Mortality in the poultry was followed for 20 weeks after the first vaccination and blood samples were taken every 4 weeks from chickens for estimation of antibodies against Newcastle disease virus. Sixty-three percent of the chicken population and 60% of the turkey population in the control village died during the 20 weeks of observation. Necropsied birds showed lesions consistent with Newcastle disease. Mortality did not exceed 22% in the vaccinated villages.     

Characterisation of an antigenically unusual virus responsible for two outbreaks of Newcastle disease in the Republic of Ireland in 1990.

Antigenic characterisation of two highly virulent virus isolates from outbreaks of Newcastle disease on two closely connected farms in County Monaghan, Republic of Ireland, in 1990 showed the viruses to be indistinguishable but unlike other Newcastle disease viruses so far tested. However, they appeared to be antigenically closest to avirulent viruses isolated from waterfowl from several countries and from chickens in Northern Ireland in 1986. Despite the antigenic differences, chickens vaccinated with a live commercial Hitchner B1 vaccine were protected against intramuscular challenge with one of the virulent isolates.     

1株广东鸡源新城疫病毒的分离鉴定和毒力基因分析

从广东粤东地区某商品蛋鸡场的发病鸡群中分离到1株病毒（YS株）,经血凝和血凝抑制试验确定为新城疫病毒（NDV）.对该分离株的F蛋白氨基酸序列的分析结果表明,其F0裂解位点的氨基酸序列为1nR-R-Q-K-R-F117,且含有101K和121V,符合典型NDV强毒株的分子特点.遗传分析结果显示分离株属于基因Ⅶd亚型.F和HN基因的氨基酸相似性分析表明,分离株与基因Ⅶ型NDV Chicken/China/Shandong/02/2010株的相似性最高,均达到99％,而与常用疫苗株B1、V4、Clone30、Mukteswar和La Sota的相似性则较低,分别在86.8％～ 90.4％和87.2％～88.3％之间,说明分离株与经典的NDV毒株存在一定的差异.     

Adequacy of commercial lentogenic vaccines against Canadian strains of viscerotropic Newcastle disease virus.

Three field strains of Newcastle Disease virus, designated S20, S21 and S23, isolated from chickens or turkeys in Ontario during the 1971-72 epizootic, were characterized as velogenic viscerotropic viruses. No significant antigenic differences were demonstrated among B1, LaSota and a field strain (S23) of velogenic vescerotropic virus by haemagglutination inhibition or protection tests. Primary water vaccination of chicks with commercial B1 and LaSota vaccines at five weeks of age and aerosol revaccination with the same strains four weeks later resulted in protection that lasted 16 weeks after revaccination against experimental challenge with strain S23. The differences in haemagglutination inhibition titres noted when the homologous or the heterologous viruses were used as haemagglutinating antigen were not statistically significant. The rates of decay of virus neutralizing and haemagglutination inhibition antibodies in vaccinated birds showed a divergence indicating the possible duality of antibodies measured in serum neutralization and haemagglutination inhibition tests.     

不同禽源的禽Ⅰ型副黏病毒株毒力、免疫原性及抗原相关性研究

从病死的鸡、鹅、鸽中分离到3株禽I型副黏病毒(CPMV、GPMV、PPMV)。毒力测定结果显示,CPMV、GPMV的毒力与鸡新城疫病毒(NDV)F48E9株的毒力相似,为强毒型;PPMV的毒力与鸡NDVLaSota株的毒力相似,为自然弱毒株。对该分离株F基因裂解位点的序列分析结果表明,CPMV、GPMVF裂解位点氨基酸序列为112K-R-Q-K-R-F117,符合强毒株的序列特征;PPMV F裂解位点氨基酸序列为112G-R-Q-G-R-L117,属于弱毒株的特征性序列。将该3株病毒制成灭活疫苗,分别免疫鸡,结果都能诱导鸡体产生较高水平的HI抗体;再分别以CPMV、GPMV攻击免疫鸡群,结果显示免疫鸡群可得到保护,而非免疫鸡群被攻击后全部致死。用HI方法检测3株病毒与Lasota株之间的交叉血凝抑制试验,结果表明LaSota与GPMV两毒株间的抗原性无明显差异,LaSota与CPMV、PPMV两毒株间的抗原性有较小的差异。本研究将有助于进一步开展禽Ⅰ型副黏病毒的致病机制和新型疫苗等方面的研究。     

从自然发病鸡中分离出一株新城疫病毒

应用电镜技术、血清学检查、生物学试验等方法,从未注射过疫苗的某自然发病鸡群分离出一株新城疫病毒。该分离株对１０日龄鸡胚的平均致死时间为１１６小时,静脉接种致病指数为０．０６,其毒力介于新城疫病毒的ＬａＳｏｔａ株与Ｖ４株之间。免疫原性试验结果表明,该分离株具有免疫后无不良反应、免疫后６天开始产生抗体、产生的抗体效价高、对新城疫强毒的攻击能１００％保护等特点,是一株良好的新城疫候选疫苗株     

Comparison of two infectious bursal disease vaccine strains: efficacy and potential hazards in susceptible and maternally immune birds.

Two infectious bursal disease vaccines were administered to separate groups of maternally immune and susceptible chickens at various ages. Vaccine B caused no damage to the bursae of chickens examined histologically at nine and 20 days after vaccination. The bursae of chickens given vaccine A were shown to be severely damaged when similarly examined. Both vaccines protected all the susceptible groups against challenge, but only vaccine A protected the groups of maternally immune chickens. Susceptible chickens vaccinated at one day of age with vaccine A showed a lowered response to Hitchner B1 Newcastle disease vaccine given at 14 days of age, judged by the haemagglutination-inhibition response and Newcastle disease challenge. The performance of the Newcastle disease vaccine was not affected in chickens given vaccine B. Bedding used by birds given vaccine A was shown to be capable of transmitting vaccinal virus to susceptible chickens, causing severe bursal damage.     

Vaccination of ostriches (Struthio camelus, Linnaeus, 1758) against Newcastle disease: evidence for vaccine compatibility and seroconversion after vaccinations using the hemagglutination inhibition and virus neutralization tests

Newcastle disease (ND) remains to be the worldwide most important infectious disease of poultry. This epizootic is in Germany and many other countries a notifiable disease. Prophylactic vaccination is the major tool for the control of ND in poultry and other birds. Eighty-three ostriches (Struthio camelus) which were kept on farms in Germany were checked for the presence of NDV-specific antibodies. Some of these birds are said to be vaccinated against Newcastle disease. Only some of these ostriches contained antibodies which were measurable in haemagglutination inhibition and virus neutralisation tests. Twenty-three previously unvaccinated ostriches were vaccinated with commercially available vaccines. Both the LaSota live and inactivated oil emulsion vaccines were well tolerated following conjunctival or subcutaneous application, respectively. Neither local nor systemic side reactions were observed. After the vaccinations high antibody titres were detected in hemagglutination inhibition and virus neutralisation tests. A strong correlation between both established methods (r = 0.92; < 0.001) were noted.     

Antibody detection-based differential ELISA for NDV-infected or vaccinated chickens versus NDV HN-subunit vaccinated chickens

With the advent of subunit vaccines for microbial diseases it is becoming increasingly important to be able to differentiate naturally infected animals from those vaccinated with the corresponding subunit vaccine. For avian viruses such as Newcastle disease virus (NDV), a whole virus-based ELISA cannot make such a differential diagnosis since in both cases the antisera would react with the whole virus. The nucleocapsid protein (NP) gene of the NDV Hitchner B1 strain was cloned, sequenced and expressed to develop a differential ELISA. The B1 NP had 95.7 and 96.1% amino acid identities with the NP of the d26 and Ulster 2C strains, respectively. The B1 NP expressed in a baculovirus expression vector (recNP) was the expected size and reacted with NDV-specific antibodies (Ab) in Western blots and by radioimmunoprecipitation. The ELISA using recNP-coated wells, tested on serum samples from flocks pretested with a commercial NDV kit gave results corresponding to those of the kit. Furthermore, use of both the renNP-based ELISA and a whole virus ELISA allowed the differentiation of birds vaccinated and a NDV haemagglutinin-neuraminidase (HN) expressing fowlpox virus from birds infected with NDV. This provides the basis for establishing an ELISA that discriminates between the antibody response to a recombinant fowlpox vaccine (expressing NDV HN protein) and that to live and inactivated NDV.     

野禽新城疫病毒F和HN基因的遗传变异趋势

野禽携带的新城疫病毒是新城疫流行传播的重要的潜在传染源.结合本实验室克隆测序的鸽NDV(PB9601)、企鹅NDV(QE01)以及GenBank下载的44株野禽(包括野鸭、鹦鹉、鸽子、天鹅、鸳鸯、雁、火鸡等)的NDV毒株的融合蛋白(Fusion gene,F)基因和血凝素蛋白(Hemagglutinin-neuraminidase gene,HN)基因分别进行了F蛋白裂解位点、基因分型以及F和HN基因氨基酸全长的遗传距离分析.结果表明:野禽感染NDV F基因的基因型以Ⅶ和Ⅵ为主,同时I、II、V和IX多种基因型并存.同一种属、同一地区的毒株间的遗传距离较近,具有明显的种属性和区域性,且绝大多数分离株与经典毒株LaSota、V4、B1、TEX-48等的遗传距离相对较远;对HN基因的遗传进化关系分析得出了相似的结论.     

一株鸭源新城疫病毒毒力基因分析及其对鸭的致病性研究

为了解中国鸭源新城疫病毒(Newcastle disease virus,NDV)的毒力特点,从2009年广东地区发病鸭群中分离和鉴定出1株新城疫病毒(简称NDV-104),对其生物学特性、致病性和融合蛋白(fusion,F)、血凝素神经氨酸酶(hemagglutinin-neuraminidase,HN)基因进行了研究。结果显示,分离株的MDT、ICPI和IVPI分别为56.4 h、1.95和1.64,结合F蛋白裂解位点(112~117位)的氨基酸序列分析,确定了分离株为新城疫强毒。致病性结果表明,分离病毒对雏鸭具有感染性和致病性。F基因遗传进化结果显示,分离株属于基因Ⅶd亚型。F、HN蛋白的氨基酸同源性结果表明,分离株与2000年以来国内外分离到的基因Ⅶ型NDV同源性较高,分别为96.6%~99.3%和97.0%~99.7%,而其与常用疫苗株B1、V4、Clone30、Mukteswar和LaSota的F、HN蛋白氨基酸序列的同源性较低,且与LaSota株和V4株的同源性最低,分别仅为88.1%和87.6%,说明分离株与经典新城疫病毒毒株存在一定的差异。     

Positive identification of Newcastle disease virus vaccine strains and detection of contamination in vaccine batches by restriction site analysis of the matrix protein gene

Twelve vaccine batches prepared from avirulent vaccine strains of Newcastle disease virus produced by seven manufacturers were identified by analysis of the matrix (M) protein gene with restriction enzymes MboI and HinfI. The analyses have revealed the presence of the strain indicated by the manufacturers (namely B-1, LaSota or Ulster 2C), except in one case when the vaccine contained strain V4 Queensland instead of VGGA as indicated. In addition, several batches of both monovalent and combined vaccines containing strain LaSota of the same company consistently disclosed contamination with strain B-1. The mixed nature of the preparations was verified not only by the dual patterns of restriction fragments but also by separating the two components and identifying them individually. Restriction analysis of the M gene, by allowing positive identification of each of the lentogenic vaccine strains, should provide an improvement in controlling vaccine batches by revealing homologous contaminants or exchange of the vaccine strain.