Newcastle Disease Virus Fusion Protein Is the Major Contributor to Protective Immunity of Genotype-Matched Vaccine

Virulent strains of Newcastle disease virus (NDV) can cause devastating disease in chickens worldwide. Although the current vaccines are substantially effective, they do not completely prevent infection, virus shedding and disease. To produce genotype-matched vaccines, a full-genome reverse genetics system has been used to generate a recombinant virus in which the F protein cleavage site has been changed to that of avirulent vaccine virus. In the other strategy, the vaccines have been generated by replacing the F and HN genes of a commercial vaccine strain with those from a genotype-matched virus. However, the protective efficacy of a chimeric virus vaccine has not been directly compared with that of a full-genome virus vaccine developed by reverse genetics. Therefore, in this study, we evaluated the protective efficacy of genotype VII matched chimeric vaccines by generating three recombinant viruses based on avirulent LaSota (genotype II) strain in which the open reading frames (ORFs) encoding the F and HN proteins were replaced, individually or together, with those of the circulating and highly virulent Indonesian NDV strain Ban/010. The cleavage site of the Ban/010 F protein was mutated to the avirulent motif found in strain LaSota. In vitro growth characteristics and a pathogenicity test indicated that all three chimeric viruses retained the highly attenuated phenotype of the parental viruses. Immunization of chickens with chimeric and full-length genome VII vaccines followed by challenge with virulent Ban/010 or Texas GB (genotype II) virus demonstrated protection against clinical disease and death. However, only those chickens immunized with chimeric rLaSota expressing the F or F plus HN proteins of the Indonesian strain were efficiently protected against shedding of Ban/010 virus. Our findings showed that genotype-matched vaccines can provide protection to chickens by efficiently preventing spread of virus, primarily due to the F protein.     

Assembly and immunological properties of Newcastle disease virus-like particles containing the respiratory syncytial virus F and G proteins

Human respiratory syncytial virus (RSV) is a serious respiratory pathogen in infants and young children as well as elderly and immunocompromised populations. However, no RSV vaccines are available. We have explored the potential of virus-like particles (VLPs) as an RSV vaccine candidate. VLPs composed entirely of RSV proteins were produced at levels inadequate for their preparation as immunogens. However, VLPs composed of the Newcastle disease virus (NDV) nucleocapsid and membrane proteins and chimera proteins containing the ectodomains of RSV F and G proteins fused to the transmembrane and cytoplasmic domains of NDV F and HN proteins, respectively, were quantitatively prepared from avian cells. Immunization of mice with these VLPs, without adjuvant, stimulated robust, anti-RSV F and G protein antibody responses. IgG2a/IgG1 ratios were very high, suggesting predominantly T(H)1 responses. In contrast to infectious RSV immunization, neutralization antibody titers were robust and stable for 4 months. Immunization with a single dose of VLPs resulted in the complete protection of mice from RSV replication in lungs. Upon RSV intranasal challenge of VLP-immunized mice, no enhanced lung pathology was observed, in contrast to the pathology observed in mice immunized with formalin-inactivated RSV. These results suggest that these VLPs are effective RSV vaccines in mice, in contrast to other nonreplicating RSV vaccine candidates.     

表达新城疫病毒F48E8株血凝素—神经氨酸酶的重组鸡痘病毒的构建 …

在克隆和鉴定新城疫病毒（ＮＤＶ）Ｆ４８Ｅ８株血凝素－神经氨酸酶（ＨＮ）基因的基础上,应用分子克隆技术将ＨＮ基因导入鸡痘病毒插入载体ｐＦＧ１１７５－１中启动子Ｐ７．５的下游,得到携带ＮＤＶ－ＨＮ基因的质粒ｐＦＧＨＮ１１７５－１。将此质粒ｐＦＧＨＮ１１７５－１以脂质体转染中国鸡痘病毒疫苗株２８２Ｅ４株感染３￣４ｈ的鸡胚成纤维细胞,采用蓝斑筛选方法纯化３次,得到稳定的重组鸡痘病毒。用ＮＤＶ－ＨＮ基因特异     

Generation by Reverse Genetics of an Effective,Stable, Live-Attenuated Newcastle Disease Virus Vaccine Based on a Currently Circulating,Highly Virulent Indonesian Strain

Newcastle disease virus (NDV) can cause severe disease in chickens. Although NDV vaccines exist, there are frequent reports of outbreaks in vaccinated chickens. During 2009–2010, despite intense vaccination, NDV caused major outbreaks among commercial poultry farms in Indonesia. These outbreaks raised concern regarding the protective immunity of current vaccines against circulating virulent strains in Indonesia. In this study, we investigated whether a recombinant attenuated Indonesian NDV strain could provide better protection against prevalent Indonesian viruses. A reverse genetics system for the highly virulent NDV strain Banjarmasin/010/10 (Ban/010) isolated in Indonesia in 2010 was constructed. The Ban/010 virus is classified in genotype VII of class II NDV, which is genetically distinct from the commercial vaccine strains B1 and LaSota, which belong to genotype II, and shares only 89 and 87% amino acid identity for the protective antigens F and HN, respectively. A mutant virus, named Ban/AF, was developed in which the virulent F protein cleavage site motif “RRQKR↓F” was modified to an avirulent motif “GRQGR↓L” by three amino acid substitutions (underlined). The Ban/AF vaccine virus did not produce syncytia or plaques in cell culture, even in the presence of added protease. Pathogenicity tests showed that Ban/AF was completely avirulent. Ban/AF replicated efficiently during 10 consecutive passages in chickens and remained genetically stable. Serological analysis showed that Ban/AF induced higher neutralization and hemagglutination inhibition antibody titers against the prevalent viruses than the commercial vaccines B1 or LaSota. Both Ban/AF and commercial vaccines provided protection against clinical disease and mortality after challenge with virulent NDV strain Ban/010 (genotype VII) or GB Texas (genotype II). However, Ban/AF significantly reduced challenge virus shedding from the vaccinated birds compared to B1 vaccine. These results suggest that Ban/AF can provide better protection than commercial vaccines and is a promising vaccine candidate against NDV strains circulating in Indonesia.     

Recombinant newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates

Paramyxoviruses such as human parainfluenza viruses that bear inserts encoding protective antigens of heterologous viruses can induce an effective immunity against the heterologous viruses in experimental animals. However, vectors based on common human pathogens would be expected to be restricted in replication in the adult human population due to high seroprevalence, an effect that would reduce vector immunogenicity. To address this issue, we evaluated Newcastle disease virus (NDV), an avian paramyxovirus that is serotypically distinct from common human pathogens, as a vaccine vector. Two strains were evaluated: the attenuated vaccine strain LaSota (NDV-LS) that replicates mostly in the chicken respiratory tract and the Beaudette C (NDV-BC) strain of intermediate virulence that produces mild systemic infection in chickens. A recombinant version of each virus was modified by the insertion, between the P and M genes, of a gene cassette encoding the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN) protein, a test antigen with considerable historic data. The recombinant viruses were administered to African green monkeys (NDV-BC and NDV-LS) and rhesus monkeys (NDV-BC only) by combined intranasal and intratracheal routes at a dose of 10(6.5) PFU per site, with a second equivalent dose administered 28 days later. Little or no virus shedding was detected in nose-throat swabs or tracheal lavages following immunization with either strain. In a separate experiment, direct examination of lung tissue confirmed a highly attenuated, restricted pattern of replication by parental NDV-BC. The serum antibody response to the foreign HN protein induced by the first immunization with either NDV vector was somewhat less than that observed following a wild-type HPIV3 infection; however, the titer following the second dose exceeded that observed with HPIV3 infection, even though HPIV3 replicates much more efficiently than NDV in these animals. NDV appears to be a promising vector for the development of vaccines for humans; one application would be in controlling localized outbreaks of emerging pathogens.     

Antigenicity and hemaglutination activity of a recombinant Hemagglutinin-neuraminidase of paramyxovirus Tianjin strain

Paramyxovirus Tianjin strain, a new genotype of Sendal virus, was isolated from the lungs of common cotton-eared marmoset that died of severe respiratory infection in the marmoset colonies. The 19.28% IgM positive rate in the young children with acute respiratory tract infection suggested a close relationship between Tianjin strain and humans. Hemagglutinin-neuraminidase (HN) is its major transmembrane glycoprotein responsible for viral attachment, penetration and release. To clear the relationship between HN structure and function of paramyxovirus Tianjin strain, rHN1, rHN2 and rHN3 overlapping the ectodomain of HN protein were expressed. Their antigenicity and hemaglutination activity, as well as cross reactivity to standard antisera against influenza virus type A, type B were analyzed. The results indicated expressed rHNs have the natural antigenicity.The segment rHN2 possesses more linear epitopes exposed on the surface of the native I-IN protein than found in segments rHN3 and rHN1. The hemagglutination activity of segment rHN3 is higher than that of segments rHN2 and rHN1, and partially dependent on the three-dimensional conformation of HN3 protein. Cross-reactivity between rHNs and standard antisera against influenza virus type A, type B suggested that rHNs might not be the best alternative as specific antigens to detect virus in clinicalserum specimens.     

Immunogenicity increase of autologous tumor cell vaccines by virus infection and attachment of bispecific antibodies

 A new type of cancer vaccine for therapeutic application in cancer patients is described. It consists of three components. (1) autologous tumor cells, (2) Newcastle Disease Virus (NDV), to be used for infection and (3) bispecific antibodies (bsAb) which attach to the viral hemagglutinin neuraminidase (HN) molecule on the infected tumor cells. A standardized procedure has been developed for generating virus infected human autologous tumor cell vaccines (ATV-NDV) which includes cell dissociation, removal of leukocytes and cell debris, gamma-irradiation and cryopreservation. Infection with the non-virulent strain NDV Ulster is performed within 30 min of co-incubation. While virus infection already increased immunogenicity of the tumor vaccine, further augmentation of T cell stimulatory capacity is achieved by attachment of specially designed bi-specific antibodies (bs HN × CD28 or bs HN × CD3). Received: 6 August 1996 / Accepted: 20 September 1996     

A recombinant Newcastle disease virus (NDV) expressing VP2 protein of infectious bursal disease virus (IBDV) protects against NDV and IBDV

Infectious bursal disease virus (IBDV) causes a highly immunosuppressive disease in chickens. Currently available, live IBDV vaccines can lead to generation of variant viruses. We have developed an alternative vaccine that will not create variant IBDV. By using the reverse genetics approach, we devised a recombinant Newcastle disease virus (NDV) vector from a commonly used vaccine strain LaSota to express the host-protective immunogen VP2 of a variant IBDV strain GLS-5. The gene encoding the VP2 protein of the IBDV was inserted into the most 3'-proximal locus of a full-length NDV cDNA for high-level expression. We successfully recovered the recombinant virus, rLaSota/VP2. The rLaSota/VP2 was genetically stable, at least up to 12 serial passages in chicken embryos, and was shown to express the VP2 protein. The VP2 protein was not incorporated into the virions of recombinant virus. Recombinant rLaSota/VP2 replicated to a titer similar to that of parental NDV strain LaSota in chicken embryos and cell cultures. To assess protective efficacy of the rLaSota/VP2, 2-day-old specific-pathogen-free chickens were vaccinated with the recombinant virus and challenged with a highly virulent NDV strain Texas GB or IBDV variant strain GLS-5 at 3 weeks postvaccination. Vaccination with rLaSota/VP2 generated antibody responses against both NDV and IBDV and provided 90% protection against NDV and IBDV. Booster immunization induced higher levels of antibody responses against both NDV and IBDV and conferred complete protection against both viruses. These results indicate that the recombinant NDV can be used as a vaccine vector for other avian pathogens.     

Prevention of disease induced by a partially heterologous AIDS virus in rhesus monkeys by using an adjuvanted multicomponent protein vaccine

Recombinant protein subunit AIDS vaccines have been based predominantly on the virus envelope protein. Such vaccines elicit neutralizing antibody responses that can provide type-specific sterilizing immunity, but in most cases do not confer protection against divergent viruses. In this report we demonstrate that a multiantigen subunit protein vaccine was able to prevent the development of disease induced in rhesus monkeys by a partially heterologous AIDS virus. The vaccine was composed of recombinant human immunodeficiency virus type 1 (HIV-1) gp120, NefTat fusion protein, and simian immunodeficiency virus (SIV) Nef formulated in the clinically tested adjuvant AS02A. Upon challenge of genetically unselected rhesus monkeys with the highly pathogenic and partially heterologous SIV/HIV strain SHIV(89.6p) the vaccine was able to reduce virus load and protect the animals from a decline in CD4-positive cells. Furthermore, vaccination prevented the development of AIDS for more than 2.5 years. The combination of the regulatory proteins Nef and Tat together with the structural protein gp120 was required for vaccine efficacy.     

Novel polyvalent live vaccine against varicella–zoster and mumps virus infections

The varicella–zoster virus (VZV) Oka vaccine strain (vOka) is a highly immunogenic and safe live vaccine that has long been used worldwide. Because its genome is large, making it suitable for inserting foreign genes, vOka is considered a candidate vector for novel polyvalent vaccines. Previously, a recombinant vOka, rvOka‐HN, that expresses mumps virus (MuV) hemagglutinin‐neuraminidase (HN) was generated by the present team. rvOka‐HN induces production of neutralizing antibodies against MuV in guinea pigs. MuV also expresses fusion (F) protein, which is important for inducing neutralizing antibodies, in its viral envelope. To induce a more robust immune response against MuV than that obtained with rvOka‐HN, here an rvOka expressing both HN and F (rvOka‐HN‐F) was generated. However, co‐expression of HN and F caused the infected cells to form syncytia, which reduced virus titers. To reduce the amount of cell fusion, an rvOka expressing HN and a mutant F, F(S195Y) were generated. Almost no syncytia formed among the rvOka‐HN‐F(S195Y)‐infected cells and the growth of rvOka‐HN‐F(S195Y) was similar to that of the original vOka clone. Moreover, replacement of serine 195 with tyrosine had no effect on the immunogenicity of F in mice and guinea pigs. Although obvious augmentation of neutralizing antibody production was not observed after adding F protein to vOka‐HN, the anti‐F antibodies did have neutralizing activity. These data suggest that F protein contributes to induction of immune protection against MuV. Therefore this recombinant virus is a promising candidate vaccine for polyvalent protection against both VZV and MuV.     

中国禽源新城疫病毒(NDV)流行株F和HN基因的遗传演化和变异频率

【目的】新城疫(ND)是中国流行最严重的疫病之一,对家禽业可造成巨大的经济损失,疫苗防控是控制ND的重要措施。新城疫病毒(NDV)流行株的遗传演化一直是研究NDV的焦点。本文利用分子信息学手段,通过比较近20年间NDV流行株不同基因型F和HN基因的分子特征和遗传变异频率,解析免疫压力下NDV的演化规律。【方法】利用Lasergene 7.1和MEGA5.1软件,选取本实验室89株NDV分离株,结合从Gen Bank下载的364株NDV流行株以及15株NDV经典毒株的基因序列,对其进行系统发育、分子特征和替代频率分析。【结果】系统发育表明,NDV已经演化为15个基因型。一致性比较显示,NDV流行株相同基因型之间核苷酸(氨基酸)高度同源,而不同基因型之间差异较大且存在明显的氨基酸变异积累。NDV基因型的分布与时间、地域密切相关,VII d亚型为中国NDV优势流行株。为评估NDV变异的频率,以Go/GD/QY/1997株(中国较早发生的基因VII亚型)为参照,1997-2015年间NDV的F/HN基因的年平均核苷酸(氨基酸)替代率为2.31×10~(-3)(2.26×10~(-3))/3.37×10~(-3)(2.35×10~(-3))。其中,1997-2001年(未使用基因VII型疫苗)F/HN基因核苷酸年平均替代率为4.72×10~(-3)/8.28×10~(-3);2002-2015年(疫苗使用后)为1.6×10~(-3)/1.84×10~(-3),显示出基因VII型疫苗在控制NDV变异速度方面具有明显的效果。【结论】生物信息学分析证实:研制出与NDV流行毒株相匹配的新型疫苗是控制当前NDV变异的关键。     

置换HN基因对新城疫病毒LaSota株致病力的影响

[目的]新城疫病毒的血凝素.神经氨酸酶(HN)和融合蛋白(F)在病毒装配、出芽、释放及侵入宿主细胞的过程中发挥关键作用,但HN对病毒致病力的影响程度尚不完全清楚.[方法]为探讨这一问题,本研究以中等毒力毒株Mukteswar的HN基因替换我国广泛应用的LaSota疫苗株HN基因,通过反向遗传操作技术拯救出嵌合病毒(rL-MuHN).[结果]rL-MuHN红细胞吸附能力较亲本株rLaSota无显著升高,具有相似的细胞融合活性;嵌合病毒ICPI由rLaSota株的0.36降为0,MDT≥90,IVPI=0与rLaSota株相同,保持典型低致病力缓发型特点不变.进一步以Mukteswar株F基因替换rL-MuHN的F基因,拯救出F和HN双基因替换嵌合病毒rL-MuFHN,尽管该病毒的细胞融合能力显著提高,但其MDT、ICPI和IVPI分别为98 h,0.59和0,显示F和HN双基因替换仍未能使嵌合新城疫病毒rL-MuFHN的致病力达到中等毒力毒株Mukteswar(MDT、ICPI及IVPI分别为46 h、1.32和0.64)的水平.[结论]试验结果表明,F及HN囊膜蛋白基因之外的病毒基因组骨架背景对病毒的致病性同样具有重要的决定性意义,不同HN蛋白对嵌合病毒的致病能力的影响不同,与供体毒株毒力无关;以流行野毒株HN替代rLaSota疫苗株构建抗原针对性更强的弱毒疫苗株存在技术可行性.     

Newcastle disease virus-vectored rabies vaccine is safe, highly immunogenic, and provides long-lasting protection in dogs and cats

Effective, safe, and affordable rabies vaccines are still being sought. Newcastle disease virus (NDV), an avian paramyxovirus, has shown promise as a vaccine vector for mammals. Here, we generated a recombinant avirulent NDV La Sota strain expressing the rabies virus glycoprotein (RVG) and evaluated its potential to serve as a vaccine against rabies. The recombinant virus, rL-RVG, retained its high-growth property in chicken eggs, with titers of up to 10^9.8 50% egg infective doses (EID₅₀)/ml of allantoic fluid. RVG expression enabled rL-RVG to spread from cell to cell in a rabies virus-like manner, and RVG was incorporated on the surface of the rL-RVG viral particle. RVG incorporation did not alter the trypsin-dependent infectivity of the NDV vector in mammalian cells. rL-RVG and La Sota NDV showed similar levels of sensitivity to a neutralization antibody against NDV and similar levels of resistance to a neutralization antibody against rabies virus. Animal studies demonstrated that rL-RVG is safe in several species, including cats and dogs, when administered as multiple high doses of recombinant vaccine. Intramuscular vaccination with rL-RVG induced a substantial rabies virus neutralization antibody response and provided complete protection from challenge with circulating rabies virus strains. Most importantly, rL-RVG induced strong and long-lasting protective neutralization antibody responses to rabies virus in dogs and cats. A low vaccine dose of 10^8.3 EID₅₀ completely protected dogs from challenge with a circulating strain of rabies virus for more than a year. This is the first study to demonstrate that immunization with an NDV-vectored vaccine can induce long-lasting, systemic protective immunity against rabies.     

Preparation and Efficacy of a Live Newcastle Disease Virus Vaccine Encapsulated in Chitosan Nanoparticles

Background

Newcastle disease (ND) is a highly contagious viral disease of poultry caused by pathogenic strains of the Newcastle disease virus (NDV). Live NDV vaccines are administered by drinking water, eyedrops or coarse aerosol spray. To further enhance mucosal immune responses, chitosan nanoparticles were developed for the mucosal delivery of a live NDV vaccine.

Methodology/Principal Findings

A lentogenic live-virus vaccine (strain LaSota) against NDV encapsulated in chitosan nanoparticles were developed using an ionic crosslinking method. Chitosan nanoparticles containing the lentogenic live-virus vaccine against NDV (NDV-CS-NPs) were produced with good morphology, high stability, a mean diameter of 371.1 nm, an encapsulation rate of 77% and a zeta potential of +2.84 mV. The Western blotting analysis showed that NDV structural proteins were detected in NDV-CS-NPs. The virus release assay results of NDV-CS-NPs indicated that NDV was released from NDV-CS-NPs. Chickens immunized orally or intranasally with NDV-CS-NPs were fully protected whereas one out of five chickens immunized with the LaSota live NDV vaccine and three out of five chickens immunized with the inactivated NDV vaccine were dead after challenge with the highly virulent NDV strain F48E9.

Conclusions/Significance

NDV-CS-NPs induced better protection of immunized specific pathogen free chickens compared to the live NDV vaccine strain LaSota and the inactivated NDV vaccine. This study lays a foundation for the further development of mucosal vaccines and drugs encapsulated in chitosan nanoparticles.     

Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations

Despite the success in expressing a variety of subunit vaccine proteins in plants and the recent stride in improving vaccine accumulation levels by transient expression systems, there is still no plant-derived vaccine that has been licensed for human use. The lack of commercial success of plant-made vaccines lies in several technical and regulatory barriers that remain to be overcome. These challenges include the lack of scalable downstream processing procedures, the uncertainty of regulatory compliance of production processes, and the lack of demonstration of plant-derived products that meet the required standards of regulatory agencies in identity, purity, potency and safety. In this study, we addressed these remaining challenges and successfully demonstrate the ability of using plants to produce a pharmaceutical grade Norwalk virus (NV) vaccine under current Good Manufacture Practice (cGMP) guidelines at multiple gram scales. Our results demonstrate that an efficient and scalable extraction and purification scheme can be established for processing virus-like particles (VLPs) of NV capsid protein (NVCP). We successfully operated the upstream and downstream NVCP production processes under cGMP regulations. Furthermore, plant-derived NVCP VLP demonstrates the identity, purity, potency and safety that meet the preset release specifications. This material is being tested in a Phase I human clinical trial. This research provides the first report of producing a plant-derived vaccine at scale under cGMP regulations in an academic setting and an important step for plant-produced vaccines to become a commercial reality.     

Evaluation of the Newcastle disease virus F and HN proteins in protective immunity by using a recombinant avian paramyxovirus type 3 vector in chickens

Newcastle disease virus (NDV) belongs to serotype 1 of the avian paramyxoviruses (APMV-1) and causes severe disease in chickens. Current live attenuated NDV vaccines are not fully satisfactory. An alternative is to use a viral vector vaccine that infects chickens but does not cause disease. APMV serotype 3 infects a wide variety of avian species but does not cause any apparent disease in chickens. In this study, we constructed a reverse-genetics system for recovery of infectious APMV-3 strain Netherlands from cloned cDNAs. Two recombinant viruses, rAPMV3-F and rAPMV3-HN, were generated expressing the NDV fusion (F) and hemagglutinin-neuraminidase (HN) proteins, respectively, from added genes. These viruses were used to immunize 2-week-old chickens by the oculonasal route in order to evaluate the contribution of each protein to the induction of NDV-specific neutralizing antibodies and protective immunity. Each virus induced high titers of NDV-specific hemagglutination inhibition and serum neutralizing antibodies, but the response to F protein was greater. Protective immunity was evaluated by challenging the immunized birds 21 days later with virulent NDV via the oculonasal, intramuscular, or intravenous route. With oculonasal or intramuscular challenge, all three recombinant viruses (rAPMV3, rAPMV3-F, and rAPMV3-HN) were protective, while all unvaccinated birds succumbed to death. These results indicated that rAPMV3 alone can provide cross-protection against NDV challenge. However, with intravenous challenge, birds immunized with rAPMV3 were not protected, whereas birds immunized with rAPMV3-F alone or in combination with rAPMV3-HN were completely protected, and birds immunized with rAPMV3-HN alone were partially protected. These results indicate that the NDV F and HN proteins are independent neutralization and protective antigens, but the contribution by F is greater. rAMPV3 represents an avirulent vaccine vector that can be used against NDV and other poultry pathogens.     

表达鸡新城疫病毒F、HN基因和传染性喉气管炎病毒gB基因的重组鸡痘病毒的构建及其特性研究

利用同源重组将新城疫病毒(NDV)的F和HN基因、传染性喉气管炎病毒(ILTV)的gB基因以及报告基因LacZ插入鸡痘病毒(FPV)的017株的复制非必需区,其中NDV的F、HN基因、ILTV的gB基因以及报告基因LacZ是在早晚期启动子LP2EP2的控制下,大肠杆菌报告基因LacZ在晚期启动子P11的控制下。经过10轮蓝斑纯化获得了包含了NDV的F和HN基因、ILTV的gB基因以及报告基因LacZ的重组鸡痘病毒,称为rFPV-F/HN/gB/LacZ。经PCR方法证明rFPV-F/HN/gB/LacZ基因组中含有NDV的F基因、HN基因和ILTVgB基因;间接免疫荧光试验和Western-blot试验表明NDV的F、HN蛋白和ILTVgB蛋白在rFPV-F/HN/gB/LacZ感染的CEF细胞中获得表达。与亲本毒相比,重组病毒在病毒的复制和致鸡胚成纤维细胞的病变方面无显著不同。这证明了在鸡痘病毒载体的一个复制非必需区可以同时插入多个禽类病原的多个外源基因,为制备多价基因工程疫苗奠定了基础。     

Construction and characterization of recombinant fowlpox virus co-expressing F and HN genes of newcastle disease virus and gB gene of infectious larygnotracheitis virus

The Fusion (F) and Haemagglutinin-Neuraminidase (HN) genes of Newcastle disease virus (NDV) and the glycoprotein B (gB) gene of infectious laryngothracheitis virus (ILTV) as well as a LacZ reporter gene were all inserted into a nonessential gene of fowlpox virus (FPV) 017 strain by homologous recombination. The NDV and ILTV genes were each under the control of a fowlpox virus immediate early/late promoter (LP2EP2), whereas the LacZ reporter gene expression cassette was regulated by a P11 late promoter. A recombinant FPV harboring the F, HN and gB genes as well as the LacZ gene, designated as rFPV-F/HN/gB/LacZ, was obtained after ten cycles of blue plaque purification. The presence of the NDV and ILTV genes was confirmed by PCR. The expression of the recombinant proteins in rFPV-F/HN/gB/LacZ was characterized by Western blot (F and gB proteins) and indirect immunofluorescence tests (F, HN and gB proteins). The results demonstrated that all four foreign proteins, which were encoded within a 10-kb gene fragment, could be expressed authentically and efficiently. Compared with the parental virus, rFPV-F/HN/gB/LacZ showed no obvious difference with respect to virus replication and cytopathogenic effects in the cell culture of chicken embryo fibroblasts (CEF). Overall, this study suggests that FPV can be a useful live virus vector for the expression of multiforeign genes against multiple avian pathogens.     

禽呼肠孤病毒感染对鸡法氏囊及免疫应答的影响

研究LY株禽呼肠孤病毒(ARV)感染1日龄SPF鸡后对法氏囊发育影响,对传染性法氏囊病毒(IBDV)、禽流感病毒(AIV)、新城疫病毒(NDV)疫苗免疫诱发的抗体的影响,及对强毒株IBDV致病作用的影响。结果表明,LY株ARV感染1日龄SPF鸡可引起法氏囊萎缩和部分淋巴细胞减少,但对增重及AIV和NDV疫苗免疫后抗体滴度却没有显著影响。ARV感染可降低弱毒IBDV疫苗免疫后的抗体反应,但对随后IBDV强毒株攻毒的抵抗力却与对照鸡无显著差异。经IBDV弱毒疫苗免疫后,再接种强毒株IBDV,不会引起死亡,但却仍能显著抑制对AIV、NDV疫苗免疫后的抗体滴度。然而,对于1~7日龄经ARV感染的鸡,IBDV强毒的这种免疫抑制作用又显著低于未经ARV感染的对照鸡。