Recombinant Newcastle Disease Virus as a Vaccine Vector

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1 Recombinant Newcastle Disease Virus as a Vaccine Vector Z. Huang, S. Elankumaran, A. Panda, and S. K. Samal 1 Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, 8075 Greenmead Drive, College Park, Maryland ABSTRACT Veterinary vaccines remained conventional for more than fifty years. Recent advances in the recombinant genetic engineering techniques brought forward a leap in designing vaccines for veterinary use. A novel approach of delivering protective immunogens of many different pathogens in a single virus vector was made possible with the introduction of a reverse genetics system for nonsegmented negative-sense RNA viruses. Newcastle disease virus (NDV), a nonsegmented negative-sense virus, is one of the major viruses of economic importance in the poultry industry throughout the world. Despite the availability of live virus vaccines of good potency, the intrinsic ability of attenuated strains to revert in virulence makes control of this disease by vaccination difficult. Armed with the knowledge of virulence factors of this virus, it is now possible to produce genetically stable vaccines and to engineer mutations that enhance immunogenicity. The modular nature of the genome of this virus facilitates engineering additional genes from several different pathogens or tumor-specific antigens to design contemporary vaccines for animals and humans. This review will summarize the developments in using NDV as a vaccine vector and the potential of this approach in designing next generation vaccines for veterinary use. (Key words: reverse genetics, RNA virus, paramyxovirus, Newcastle disease virus, vectored vaccine) 2003 Poultry Science 82: INTRODUCTION The use of vaccines against infectious diseases has been one of the true success stories of modern medicine. Vaccines are the most effective and inexpensive prophylactic tools in veterinary medicine. Traditionally, there are two major strategies for the production of viral vaccines: one employing modified live attenuated virus and the other employing chemically inactivated virus. However, vaccines produced by conventional means are imperfect in many respects with regard to safety, efficacy, and cost. Differentiating vaccinated animals from their infected (DIVA) counterparts is another major constraint in using live attenuated vaccines and may cause problems in disease control measures. The unsatisfactory efficacy, safety, antigenic variation, and the emergence of new diseases dictate the need to develop newer and more effective vaccines. This is particularly true for developing vaccines against poultry diseases, in which cost constraint and the need to vaccinate en masse are primary concerns. Through the use of recombinant DNA technology, it is now possible to generate live virus vaccines that promise significant improvements in safety, efficacy, and cost. By introducing multiple gene deletion mutations in the genome of a virus, it is possible to generate a new class of attenuated vaccines that are safe and may not result in reversion to virulence. Recombinant DNA technology has also made it possible to generate vaccines utilizing viruses as vectors for the expression of protective antigens of other viruses. This new class of vaccine is called vectored vaccine. Vectored vaccines are also advantageous in that DIVA is made easier by using marker genes. Although there are numerous examples of vectored vaccines reported in literature, few have been investigated as veterinary vaccines (Sheppard, 1999). To date, only three viral vector vaccines, all of which are based on pox virus vectors, have been licensed for commercial use (Sheppard, 1999). The only poultry vaccine to be licensed for use is a fowl pox virus vectored vaccine carrying Newcastle disease virus genes (Boyle and Heine, 1993; McMillen et al., 1994). Recently, recombinant replication-defective Semliki forest virus vectored vaccine against infectious bursal disease virus of chickens has been reported to produce a strong antibody response, but the efficacy of the vaccine after challenge is unknown (Phenix et al., 2003 Poultry Science Association, Inc. Received for publication August 28, Accepted for publication December 20, To whom correspondence should be addressed: ss5@umail. umd.edu. Abbreviation Key: CAT = chloramphenicol acetyl transferase; DIVA = differentiating vaccinated from infected animals; HEp-2 = human epidermoid carcinoma; HPIV 3 = human parainfluenza virus type 3; IBV = infectious bronchitis virus; NDV = Newcastle disease virus; NSV = nonsegmented negative-sense virus; rndv = recombinant NDV; RNP = ribonucleoprotein. 899

2 900 HUANG ET AL. 2001). Several studies in recent years highlighted the potential of Newcastle disease virus (NDV) to be used as a vaccine vector for avian diseases (Krishnamurthy et al., 2000; Huang et al., 2001; Nakaya et al., 2001). This review will focus on the methodologies employed and success achieved for this approach and analyze the means of improving NDV-vectored vaccines to prevent diseases of poultry. NDV Newcastle disease virus causes a highly contagious and fatal disease affecting all species of birds. The disease can vary from clinically inapparent to highly virulent forms, depending on the virus strain and the host species. The continuous spectrum of virulence displayed by NDV strains enabled the grouping of them into three different pathotypes: lentogenic, mesogenic, and velogenic (Alexander, 1997). Lentogenic strains do not usually cause disease in adult chickens and are widely used as live vaccines in poultry industries in the United States and other countries. Viruses of intermediate virulence are termed mesogenic, while viruses that cause high mortality are termed velogenic. The disease has a worldwide distribution and remains a constant major threat to commercial poultry production. Newcastle disease virus is a member of the genus rubulavirus in the family Paramyxoviridae (Pringle, 1997). The genome of NDV is a nonsegmented, single-stranded, negative-sense RNA of 15,186 nucleotides (Krishnamurthy and Samal, 1998; Phillips et al., 1998; de Leeuw and Peeters, 1999; Nakaya, et al., 2001), which is the multiple of six (Calain and Roux, 1993). The genomic RNA contains a3 leader sequence of 55 nucleotides and a 5 trailer sequence of 114 nucleotides. The leader and trailer sequences are essential for virus transcription and replication, and they flank six structural genes in the order of 3 - NP-P-M-F-HN-L-5, which encode at least seven proteins (Peeples, 1988; Steward, et al., 1993). The nucleocapsid protein (NP) binds to the genomic RNA forming the nucleocapsid core, and the phosphoprotein (P) and the large polymerase protein (L) are associated with the nucleocapsid core. Together, they form a tight functional ribonucleoprotein (RNP) complex, both in virions and infected cells. The matrix protein (M) forms the inner layer of the envelope and is probably a driving force in viral assembly. Two viral glycoproteins are present in the viral envelope: hemagglutinin-neuraminidase protein (HN) and fusion protein (F). The former is responsible for the attachment of the virus to host cell receptor, whereas the latter mediates fusion of the viral envelope with the hostcell membrane, enabling viral entry into the cell. Two additional proteins, V and W, are produced by RNA editing (Curran et al., 1991) during P gene transcription, and their functions remain unknown (Steward et al., 1993, 1995). At the beginning and end of each gene are conserved transcriptional control sequences known as genestart (3 -UGCCCAUCU/CU-5 ) and gene-end (3 -AAU/ CUUUUUU-5 ) signals. Between the gene boundaries are intergenic regions, which vary in length from 1 to 47 nucleotides (Chambers et al., 1986; Krishnamurthy and Samal, 1998). The intergenic sequences are not transcribed into mrna (Peeples, 1988). NDV follows the same general mode of transcription and replication as other nonsegmented negative-sense RNA viruses (Peeples, 1988; Pringle, 1997; Lamb and Kolakofsky, 2001). Like other nonsegmented negative-sense viruses (NSV), there is a polar attenuation of transcription such that each downstream gene is transcribed less than its upstream neighbor (Peeples, 1988; Nagai, 1999). REVERSE GENETICS SYSTEMS FOR NONSEGMENTED NEGATIVE-SENSE RNA VIRUSES The development of methods to recover NSV entirely from cloned cdna, established in recent years, opened up the possibility of genetically manipulating this virus group, including NDV (Conzelmann, 1998; Roberts and Rose, 1998). This unique molecular genetic methodology, termed reverse genetics, provides a means not only to investigate the functions of various virus-encoded genes (Palese et al., 1996; Nagai, 1999) but also to allow the use of these viruses to express heterologous genes (Bukreyev et al., 1996; Mebatsion et al., 1996; Schnell et al., 1996; Hasan et al., 1997; He et al., 1997; Sakai et al., 1999). This provides a new method of generating improved vaccines and vaccine vectors. Rabies virus was the first NSV to be recovered entirely from cloned cdna. The strategy used to recover rabies virus was to transfect cells with four plasmids encoding the full-length antigenomic viral RNA, the rabies virus nucleoprotein (N), and RNA polymerase proteins (L and P), all under the transcriptional control of a T7 RNA polymerase promoter. The T7 RNA polymerase was provided by a recombinant vaccinia virus vector (VvT7). This transfection resulted in the encapsidation of the antigenomic RNA by the N protein to form RNP and in the replication and transcription of these RNP by the viral RNA polymerase, leading to the generation of recombinant rabies virus. Reverse genetics systems now exist for numerous nonsegmented and segmented negative-sense RNA viruses, including NDV (Schnell et al., 1994; Garcin et al., 1995; Collins et al., 1995; Lawson et al., 1995; Radecke et al., 1995; Whelan et al., 1995; Bridgen and Elliot, 1996; Kato et al., 1996; Palese et al., 1996; Baron and Barrett, 1997; He et al., 1997; Hoffman and Banerjee, 1997; Jin et al., 1998; Leyrer et al., 1998; Buchholz et al., 1999; Fodor et al., 1999; Neumann et al., 1999; Peeters et al., 1999; Römer-Oberdörfer et al., 1999; Krishnamurthy et al., 2000; Huang et al., 2001). However, it should be noted that recovery of recombinant virus is governed by the individual concentrations of each expression plasmid, and in some instances as in pneumoviruses, an additional viral protein is required to be coexpressed for efficient recovery (Collins et al., 1995). While the use of VvT7 facilitated the consistent recovery of recombinant RNA viruses, it entailed several disadvantages, such as interference of

3 ANCILLARY SCIENTISTS SYMPOSIUM 901 vaccinia virus in differentiating cytopathic effects due to the recombinant NSV, the necessity to purify the recovered virus from vaccinia virus (Schnell et al., 1994; Collins et al., 1995; Whelan et al., 1995), and RNA recombination induced by vaccinia virus during recovery (Garcin et al., 1995). To circumvent these difficulties, host-restricted vaccinia virus vectors, such as vaccinia virus Ankara (MVA) strain (He et al., 1997; Leyrer et al., 1998) or cell lines constitutively expressing T7 polymerase are being currently used (Radecke et al., 1995; Schneider et al., 1997; Buchholz et al., 1999; Finke and Conzelmann, 1999; Römer-Oberdörfer et al., 1999; Harty et al., 2001). The authenticity of the recovery system for NSV from cloned cdna is now well established (Conzelmann, 1998; Roberts and Rose, 1998). Recovery systems for recombinant NSV are now based on intracytoplasmic reconstitution of the RNP complex, which represents the template for the viral polymerase and is the prerequisite for initiating an infectious cycle (Palese et al., 1996; Conzelmann, 1998; Roberts and Rose, 1998). RECOVERY SYSTEMS FOR NDV The first recovery systems, based on a lentogenic vaccine strain (LaSota) of NDV, were reported simultaneously by two independent groups in 1999 (Peeters et al., 1999; Römer-Oberdörfer et al., 1999). In the first reported system, the full-length NDV cdna from LaSota strain (ATCC-VR699) was assembled in poltv5 transcription vector containing a T7 DNA-dependent-RNA polymerase promoter with two G residues, followed by two unique restriction sites containing the full-length clone, the autocatalytic ribozyme from hepatitis delta virus, and the transcription termination signal from bacteriophage T7. Individual clones of the NDV transcriptase complex (NP, P, and L) were cloned in a eukaryotic expression vector. To generate infectious NDV, primary chicken embryo fibroblast cells or QM 5 cells were infected with fowl pox virus-t7 recombinant and cotransfected with the fulllength clone of NDV and the support plasmids. After incubation for 3 to 6 d in medium containing 5% allantoic fluid, the recombinant virus harvests from these cells were amplified in 9-to-11-d-old embryonated specific pathogen-free eggs. The use of allantoic fluid in the medium was to supply the necessary proteases for the cleavage of F protein. Creation of genetic tags in the form of additional nucleotides after L gene or changing the F cleavage site into a consensus cleavage site of virulent strains did not affect recovery of infectious virus. The cotransfection protocol for generating recombinant NDV (rndv) was claimed to be efficient in that it can generate several infective centers in infected monolayers (Peeters et al., 1999). The second system reported for recovery of a lentogenic NDV from cloned cdna essentially used the same strategy of assembling the full-length antigenomic expression plasmid and support plasmids (Römer-Oberdörfer et al., 1999). However, this system made use of BHK 21 cells, clone BSR T7/5, stably expressing the T7 RNA polymer- FIGURE 1. The reverse genetics protocol employed for generating recombinant Newcastle disease viruses (NDV). The full-length antigenomic cdna of NDV and the support plasmids encoding NP, P, and L proteins were transfected into human expidermoid carcinoma (HEp-2) cells that were preinfected with recombinant vaccinia virus (MVA), to recover the recombinant virus. The recombinant virus was then amplified in embryonated chicken eggs post-transfection. ase, as against the fowl pox virus-t7 vector in the previous system. There were three G residues inserted in the T7 promoter, preceding the first nucleotide of the leader region of the cloned NDV to enhance T7 polymerasedriven transcription. The recombinant virus produced in the cotransfected cells was amplified in embryonated chicken eggs. This system also employed unique restriction sites as genetic tags to differentiate the wild type and recombinant NDV. Although the biological properties of the cloned virus were essentially similar to that of the parental virus in both systems, the virulence of the rndv was lower than the wild-type virus in the first system. This system, in principle, exploited a constitutive cellular T7 expression system and a virus propagation step, allowing amplification of a few primary infectious recombinant virions originating from single recovery events and dispensed with the disadvantages associated with the use of recombinant vaccinia virus (Römer-Oberdörfer et al., 1999). A third system was developed recently to recover a lentogenic LaSota strain of NDV (Huang et al., 2001). This system utilized a host-restricted recombinant vaccinia virus (MVA) and human epidermoid carcinoma (HEp-2) cells for transfection, followed by amplification of the rndv in embryonated eggs. HEp-2 cells were used, as these cells are host restricted for vaccinia virus and are resistant to vaccinia virus induced cytopathic effects. To circumvent the requirement of proteases for the cleavage of F protein, medium-containing acetyl-trypsin was employed posttransfection (Figure 1). Although the method of construction of the full-length antigenomic cdna was essentially similar to the other systems, a new cistron to encode the chloramphenicol acetyl transferase (CAT) reporter gene, flanked by NDV gene-start and gene-end sequences, was incorporated before the NP open-reading frame to assess the expression of a foreign gene in the rndv. The biological properties and virulence of the rndv was reported to be similar to that of the parental strain. The only system available for the recovery of recombinant mesogenic NDV was described by Krishnamurthy et al. (2000). This system utilized the vaccinia virus recom-

4 902 HUANG ET AL. binant (MVA) and HEp-2 cells for transfection. The fulllength clone of the mesogenic strain Beaudette C and the support plasmids (N, P, and L) from the same strain were used for transfection. Two unique restriction sites were introduced as genetic markers in the full-length cdna clone. An additional transcriptional unit encoding the CAT reporter gene was placed between the HN and L genes. The growth of the rndv expressing the CAT gene was delayed and the virus was attenuated. The CAT reporter gene was stably expressed for several passages in cell culture. EXPRESSION OF FOREIGN GENES The modular nature of NSV genomes facilitates engineering additional genes with ease. The construction of viral genomes expressing foreign genes depends on the addition of the foreign gene flanked by appropriate transcription start and stop signals, which are recognized by the viral RNA-dependent-RNA polymerase. However, the level of expression of foreign genes is determined by the site of insertion within the genome, due to polar attenuation of transcription (Roberts and Rose, 1998). A variety of reporter genes as additional transcriptional units have been inserted in appropriate genome positions of several NSV, and their applications explored (Collins et al., 1991; Bukreyev et al., 1996; Mebatsion, et al., 1996; Schnell et al., 1996; Hasan et al., 1997). Genes encoding other viral antigens have been investigated in vesicular stomatitis virus (VSV) (Kretzschmar et el., 1997; Roberts et al., 1998), rinderpest virus (Baron et al., 1999), and NDV (Krishnamurthy et al., 2000; Huang et al., 2001; Nakaya et al., 2001). Chimeric viruses with replacement of genes, such as human parainfluenza virus 3 (HPIV3) and measles virus or respiratory syncytial virus and HPIV 3 recombinants have been developed to function as dual vaccines (Durbin et al., 2000; Schmidt et al., 2001). Moreover, genes that encode proteins with known antiviral activities or with known immunoregulatory functions could be inserted into the viral genome, which may attenuate the virus but augment immunogenicity. For respiratory syncytial virus (RSV), insertion of the interferon-γ gene attenuated the virus while inducing a vigorous humoral immune response (Bukreyev et al., 1999). We reported the recovery of a recombinant NDV strain Beaudette C expressing CAT reporter gene between the HN and L genes (Krishnamurthy et al., 2000). The inserted CAT gene was maintained very stably and expressed well for serials of passage in cell culture. However, the recombinant virus was associated with reduced plaque size, slower replication kinetics, and more than 100-fold decrease in yield compared to the wild-type virus. Pathogenicity studies also suggested that the recombinant virus was attenuated, most probably due to the disruption of NP:P:L ratio, which seems to be critical to viral replication. Although the results showed that the recombinant NDV could be used as a vaccine vector, they raised concerns about the level of expression of foreign gene and growth retardation of the virulent strain after insertion FIGURE 2. Comparison of chloramphenicol acetyl transferase (CAT) expression by rbc/cat and rlasota/cat viruses at the sixth passage in DF1 (chicken embryo fibroblast cell line). The CAT gene was inserted between HN and L genes in rbc/cat, while it was expressed from the 3 proximal position in rlasota/cat. Equal amounts of cell lysates were analyzed for acetylation of [ 14 C]-chloramphenicol as visualized by thin-layer chromatography. The relative CAT activity was quantified by densitometry. of the foreign gene. To address these concerns, we introduced the CAT gene into the most 3 -proximal locus of NDV strain LaSota and recovered the recombinant virus (Huang et al., 2001). With foreign gene inserted upstream of NP, a higher-level expression of the inserted gene was obtained (Figure 2). Moreover, the growth characteristics of this recombinant virus in cell culture and in vivo was similar to that of the wild-type virus (Figure 3). These results indicate that NDV can be engineered to express Virus titer (log pfu/ml) LaSota rlasota rlasota/cat Time Post-infection (h) FIGURE 3. Multistep growth curve of rlasota in DF1 cells. The chicken embryo fibroblast cells were infected with PFU per cell with three replicates per virus. Samples were collected at every 8 h for 56 h. The virus in the supernatant was titrated by a plaque-forming assay in DF1 cells. The mean virus titer + standard deviation is shown for different time points.

5 ANCILLARY SCIENTISTS SYMPOSIUM 903 foreign protein stably and manipulated for use as a vaccine vector. A full-length cdna clone of NDV vaccine strain Hitchner B1, expressing an influenza virus hemagglutinin protein, was recently constructed (Nakaya et al., 2001). In this report, the HA protein open-reading frame was inserted between the P and M genes. It was demonstrated that the rescued virus stably expressed the HA protein on the surface of infected cells and that a significant amount of the expressed protein was incorporated into the virions. The maximal titer of the recombinant virus was approximately 20-fold lower than that of the wild-type virus; however, it induced a strong protective antibody response against influenza virus in mice. As vaccination against NDV is a common practice in the poultry industry, the results obtained here suggested that the recombinant rndv vaccine strain expressing an avian influenza virus HA could have a practical use as a combined vaccine against both avian influenza viruses and NDV. ROLE OF NDV AS A VACCINE VECTOR The use of a viral vector for immunization has the general advantage that the foreign antigens are expressed naturally in the context of an infected cell, thereby inducing cellular, as well as humoral, immune responses. Therefore, the recombinant viral-vectored vaccines hold many promises for the future. In the poultry industry, so far, fowl pox virus, herpesvirus of turkeys, Marek s disease virus, adenovirus, and members of retrovirus family have been used most extensively as expression vectors (Dodds et al., 1999; Jackwood, 1999). These recombinant viruses demonstrated protective efficacy against a variety of avian diseases. However, their practical usage still needs to be evaluated, particularly with regard to factors, such as safety, efficacy, and cost of production (Yokoyama et al., 1997; Jackwood, 1999). Certain characteristics of NDV suggest that rndv expressing a foreign viral protein would be very good vaccine candidates. NDV grows to very high titers in many cell lines and eggs, and it elicits strong humoral and cellular immune responses in vivo. NDV naturally infects via respiratory and alimentary tract mucosal surfaces, so it is especially useful to deliver protective antigens of respiratory disease pathogens. In addition, commercially available live NDV vaccines are widely used in the United States and most other countries. Vaccines based on live NDV recombinant would also have advantages over other live recombinant vaccine vectors. First, the foreign protein is expressed with only a few NDV proteins. In contrast, pox and herpes virus vectors express a large number of additional proteins from their large-size genomes. For the generation of specific immune responses in vaccine applications, it would be advantageous to have only a limited number of proteins expressed. Second, NDV replicates in the cytoplasm of the infected cells without a DNA phase, which eliminates the problem of integration of viral genome into the host cell DNA. The virus does not undergo detectable genetic recombination, which would make this expression vector much more stable and safe. The availability of the reverse genetics system would greatly enhance the development of NDV as a better vaccine and vaccine vector. One great promise is the generation of novel deletion mutants. NDV encodes nonessential proteins, V and W, by way of RNA editing during the transcription of its P gene. The main function of these accessory proteins is to facilitate virus replication in vivo, by acting as an interferon antagonist (Z. Huang et al., unpublished data). Deletion of these proteins would render the virus more attenuated but still immunogenic; therefore, it could be used for in ovo vaccination (Mebatsion et al., 2001). Another application of reverse genetics is to develop a more effective and better NDV vaccine. The current NDV vaccines utilize naturally occurring lentogenic strains, such as the Hitchner B1 and LaSota. As encountered with any other live attenuated vaccines, the current NDV vaccines may still cause disease due to reversion to virulence. A highly stable and efficacious vaccine, therefore, becomes a desired objective. The introduction of the reverse genetics system to the molecular biology of NDV has opened new horizons in the development of stable NDV vaccines. For example, mutations can be introduced into a vaccine strain at the F cleavage site to prevent its reversion to virulent virus, thus making the vaccine more stable. Lentogenic vaccine strains of NDV with rearranged gene order may provide a rational strategy for vaccine development, since the recombinant virus would be more immunogenic and more attenuated at the same time, as shown in the case of vesicular stomatitis virus (Wertz et al., 1998). A recombinant chimeric NDV vaccine that allows serological differentiation between vaccinated and infected animals has been generated (Peeters, et al., 2001). In this marker virus, the HN gene has been replaced by a hybrid HN gene with immunogenic globular domain of HN from avian paramyxovirus type 4; thus, it induces a protective immune response against NDV by eliciting antibodies against the F protein but can be differentiated from NDV infection on the basis of different antibody profiles against their HN proteins. These examples show that viral attenuation through specific mutations has obvious practical significance in vaccine development. Armed with the knowledge of basic studies on the virus, genetically stable, safe, and effective NDV vaccines can be generated. Recent studies have demonstrated the potential of NDV as a vaccine vector (Krishnnamurthy et al., 2000; Huang et al., 2001; Nakaya et al., 2001). The results of these studies have shown that the expression levels of foreign proteins are quite high and the foreign genes are very stable after many passages in vitro and in vivo. It was also demonstrated that when inserted into the most 3 - proximal locus of the viral genome, the expression level of the foreign gene was higher. Moreover, the replication and pathogenesis of the recombinant virus was not modified significantly. It would be very appealing to construct a rndv to deliver the immunogenic proteins of other

6 904 HUANG ET AL. respiratory viruses, such as the spike protein of infectious bronchitis virus (IBV), due to the following considerations: both NDV and IBV infect the respiratory tract; in practice, NDV vaccines are already routinely administered as combination products containing live IBV vaccines, and the current IBV vaccine itself contributes to generation of new variants due to recombination and mutations. Therefore, NDV-vectored IBV vaccine will eliminate the generation of new variants and will aid in the control of IBV. rndv can also be used for the expression of protective antigens of avian pneumovirus, since avian pneumovirus causes severe economic losses and, currently, no effective vaccines are available. It is also highly possible to use NDV to express protective immunogens of other avian viral pathogens as polyvalent vaccines. Other genes coding for cytokines or other immunoregulatory proteins or both can also be inserted into the recombinant virus, thus, making the virus more attenuated but still highly immunogenic. Besides being a vector for polyvalent vaccines, NDV can be engineered as a surrogate virus in which the viral envelope can be completely replaced with other viral envelope proteins or by chimeric envelope proteins (Collins et al., 1999). Since the surrogate virus may be used to express other virus envelope or cellular membrane proteins to manipulate host range or cell tropism, it has a great potential for gene therapy or treatment of cancer or to prevent diseases for which no effective vaccines are available. By expressing the human coreceptor complex for HIV-1, the engineered Rhabdoviruses deliver genes only to HIV-1-infected cells, not to the normal bystander cells, as shown for rabies virus and vesicular stomatitis virus (Mebatsion et al., 1995, 1997; Schnell et al., 1997). NDV strains can infect many different types of cells, even nondividing cells, such as neurons, and certain strains of NDV can selectively kill human tumor cells (Reichard et al., 1992). Therefore, NDV would be a better choice for targeted gene expression and somatic gene therapy. Now, there is an increasing interest in the use of NDV for cancer therapy (Nelson, 1999), due to its unique properties: it binds specifically and replicates selectively in tumor cells. Thus, NDV may be exploited to treat cancer by the temporal expression of anti-angiogenic factors or the induction of immune responses to tumor-specific antigens. In conclusion, vaccination strategies have undergone a radical change in recent years. Virus vectors have been widely studied for vaccination and for use as a system of gene transfer into the living body. The use of biotechnology to create recombinant viral-vectored vaccines holds many promises for the future. With the advent of the reverse genetics system, the manipulation of NDV is as easy as that of other DNA and positive-sense RNA viruses. The modular nature of transcription, undetectable rate of recombination, and the lack of DNA phase in the replication cycle render NDV as a suitable substrate for the rational design of live attenuated vaccines and vaccine vectors. A deeper understanding of NDV molecular biology may not only be subsequently applied to the control of Newcastle disease and other related diseases in poultry and beyond through successful recombinant vaccines but may also be exploited for gene therapy and cancer treatment. ACKNOWLEDGMENTS We thank Daniel D. Rockemann and Peter Savage for excellent technical assistance. This work was supported by U.S. Department of Agriculture NRI Grant # REFERENCES Alexander, D. J Newcastle disease and other avian Paramyxoviridae infection. Pages in Diseases of Poultry. B. W. Calnek, H. J. Barnes, C. W. Beard, L. R. Mcdougald, and Y. M. 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