Newcastle disease virus - Taylor & Francis Online

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Avian Pathology ( 2001 ) 30, 439– 455

REVIEW ARTICLE

Newcastle disease virus: macromolecules and opportunities Khatijah Yusoff* & Wen Siang Tan Department of Biochemistry and Microbiology, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor D.E., Malaysia

Over the past two decades, enormous advances have occurred in the structural and biological characterization of Newcastle disease virus ( NDV). As a result, not only the complete sequence of the viral genome has been fully determined, but also a clearer understanding of the viral proteins and their respective roles in the life cycle has been achieved. This article reviews the progress in the molecular biology of NDV with emphasis on the new technologies. It also identifies the fundamental problems that need to be addressed and attempts to predict some research opportunities in NDV that can be realized in the near future for the diagnosis, prevention and treatment of disease( s).

Introduction Newcastle disease virus ( NDV) or avian paramyxovirus 1 ( Alexander, 1997) is an economically important avian virus that may result in substantial loss to the poultry industry. It has a wide host range, infecting 27 of the 50 orders of birds ( Jørgensen et al., 1998; Kuiken et al., 1998; Schelling et al., 1999; Alexander, 2000). The virus is transmitted by ingestion or inhalation and produces a disease of variable clinical severity and transmissibilit y depending on its pathotype. Based on the severity of the disease, NDV can be grouped into three pathotypes: the lentogenic strains cause only clinically mild or unapparent respiratory disease; the mesogenic strains produce respiratory and nervous signs with moderate mortality; and the viscerotropic or neurotropic velogenic strains cause severe intestinal lesions or neurological disease, resulting in high mortality ( up to 100% in chickens) ( Alexander, 1989, 1997). Although the virus is currently controlled effectively by vaccination and mass slaughtering, it remains a potential threat to commercial or backyard production, as was proven by the recent outbreaks in Australia ( Westbury, 2001) and Malaysia, resulting in mass eradication. NDV is classified as a member of the order Mononegavirales, family Paramyxoviridae, subfamily Paramyxovirinae. It was initially considered

as the prototype for the genus Paramyxovirus, but in 1993 it was placed within the genus Rubulavirus ( Rima et al., 1995 ). Since the virus did not contain the small hydrophobic gene ( Lamb & Kolakofsky, 1996 ) that is present in all of the other rubulaviruses, de Leeuw & Peeters ( 1999 ) suggested it be placed as a separate member of the Paramyxovirinae . This paper reviews the current knowledge in the molecular biology of NDV, with particular emphasis on the developments made in our laboratory over the past 10 years. The reader is referred to other reviews for more comprehensive coverage on the different aspects of the virus ( Lamb & Kolakofsky, 1996; Sharma, 1999; Alexander, 2000, 2001; Sinkovics & Horvath, 2000; Aldous & Alexander, 2001). Virion structure Electron microscopic examinations of purified preparations of NDV from infected allantoic fluid of domestic fowl embryos reveal pleomorphic structures ( Figure 1a). Most of these are roughly spherical with diameters around 100 to 500 nm. Occasionally, filamentous particles of approximately 100 nm in diameter and variable length can be seen. The virion is enveloped with a lipid bilayer membrane derived from the host cell membrane ( Figure 2a). Embedded in the envelope are two

*To whom correspondence should be addressed. E-mail: [email protected] y ISSN 0307-9457 ( print)/ISSN 1465-3338 ( online)/01/050439-1 7 DOI: 10.1080/03079450120078626

© 2001 Taylor & Francis Ltd

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Figure 1. ( a) Electron micrograph of NDV particles purified from allantoic fluid. ( b) A partly disrupted NDV particle exposing the nucleocapsid. The bars represent 100 nm.

Figure 2. ( a) Schematic representation of the virion structure of NDV. The representation of the proteins has no quantitative or positional significance. Magnification of a portion of the virion is indicated by a circle on the right; C and N, respectively, represent the carboxy-terminal end of HN and the amino-terminal end of F that are exposed on the surface of the virion. ( b) NDV genome organization and the viral transcripts. The minus strand of NDV RNA is represented by a bar in the 39 to 59 direction. The approximate length ( kb) of each gene is shown above the bar, and the number of nucleotide( s) in the intergenic leader ( l) and trailer ( t) regions is shown below the bar. Major viral transcripts are indicated as bold lines below the bar. d The mRNA start site; AAA, the polyadenylation site.

Newcastle disease virus

different glycoproteins, the haemagglutinin-neu raminidase ( HN) and fusion ( F) proteins, which appear as tiny spikes projecting from the external surface of the membrane when observed under an electron microscope. These relatively complex proteins interact with each other and are involved in viral infectivity and virulence ( Stone-Hulslander & Morrison, 1997) . Either of these proteins can induce protective immunity ( Meulemans et al., 1986; Nagy et al., 1991 ). Beneath this lipid membrane is a layer of relatively hydrophobic non-glycosylated matrix ( M) protein, which is not only associated with the membrane but also with the N-terminal segment of the HN protein located in its inner surface ( GarciaSastre et al., 1989 ). The M protein is believed to interact with the nucleocapsid ( NP) that resembles the classical herringbone morphology that can be clearly seen when the viral membrane is removed or disrupted ( Figure 1b). This herringbone-like structure comprises thousands of NP subunits that are associated tightly with several copies of phosphoprotein ( P) and large protein ( L). The non-segmented, single-stranded negative-sense RNA genome of 15 186 bases ( Krishnamurthy & Samal, 1998; Phillips et al., 1998, de Leeuw & Peeters, 1999 ) is located in the central hollow of the herringbone-like nucleocapsid. Together, these three RNA-associated proteins and the RNA genome constitute the viral transcriptase complex. The interactions between these macromolecules and their binding domains have yet to be defined completely. Viral genome The RNA genome ( Figure 2b) contains six major genes that encode the structural proteins in the order 39 -NP-P-M-F-HN-L-5 9 ( Chambers et al., 1986a; Wilde et al., 1986 ) as well as two non-structural proteins, W and V. These non-structural proteins may result from differential initiation ( McGinnes et al., 1988 ) or transcriptional editing of the P gene mRNA ( Steward et al., 1993 ). RNA sequencing studies revealed that the 39 and 59 ends of the genomic RNA respectively contain a leader sequence and a trailer sequence of some 50 nucleotides ( Kurilla et al., 1985; Yusoff et al., 1987) . Transcription of the leader sequence results in the production of the leader transcript, which is the smallest but most abundant mRNA ( Peeples, 1988). Recently, Phillips et al. ( 1998 ) showed that the trailer region of Beaudette C has 114 nucleotides. The 59 terminus of the trailer sequence shares a high degree of complementarity with that of the 39 terminal leader sequence ( Peeters et al., 2000 ). These sequences may be involved in the regulation of NDV replication, transcription and encapsidation of the genomic and antigenomic RNAs ( Lamb & Kolakofsky, 1996) . The six structural genes are separated by intergenic regions of variable lengths ( 1 to 47 nucleotides; Figure 2b) , which could be involved in terminating mRNA transcription from the preceding

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gene, before initiating transcription of the subsequent gene ( Ishada et al., 1986; Millar et al., 1986; Yusoff et al., 1987; Phillips et al., 1998 ). At least eight species of mRNA transcripts ( Figure 2b) are produced by the viral transcriptase complex, comprising the RNA genome, NP, P and L proteins. The largest of all, the genome complement RNA of the positive sense or antigenome, serves as the template for the synthesis of fulllength genomic RNA ( Duesberg & Robinson, 1965 ). Direct transcription of the start signals ( 39 -UGCCCAUCU/CU-59 ) preceding the six major genes results in six mRNAs, which are capped, methylated and terminated at a common polyadenylation site ( 39 -AA/UCUUUUUU-59 ) ( Ishada et al., 1986; Millar et al., 1986; Yusoff et al., 1987 ). Unlike these mRNAs, the leader transcript is not capped, methylated or polyadenylated ( Peeples, 1988 ). Transcriptional modification of the P gene mRNA, by insertion of one or two non-templated G residues at nucleotide position 484 in the P transcript, produces extra species of modified mRNAs ( Steward et al., 1993; Locke et al., 2000 ) that encode non-structural proteins, V ( + one nucleotide frameshift) and W ( + two nucleotide frameshifts ), respectively. Nucleocapsid protein Isolated nucleocapsids of NDV appear in negativestaining electron microscopy as flexible helical structures with a diameter of about 18 nm and 1 m m in length ( Figure 3) . Apparently, these structures resemble the classical herringbone morphology with spikes protruding from a central channel. The essential subunit of the structures is a single polypeptide of 489 residues with a molecular weight

Figure 3. An electron micrograph of the NDV nucleocapsid. The nucleocapsid resembling a herringbone morphology was isolated from NDV particles. The bar represents 100 nm.

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of about 53 kDa. Our recent studies have shown that several NP monomers form a ring-like particle and many of these particles assemble to form a fulllength nucleocapsid ( Kho et al., 2001b ). The viral RNA is located inside the central channel, surrounded by 2200 to 2600 NP subunits ( Choppin & Compans, 1975) that protect it from nuclease activities. In association with the L and P proteins, the NP protein is thought to be involved in replication and transcription of the viral genome, but neither the role of the NP protein nor the mechanisms of these processes have been investigated in depth. The NP protein has been expressed in baculovirus ( Errington & Emmerson, 1997) and Escherichia coli ( Kho et al., 2001b ), where they assemble into structures morphologically similar to authentic nucleocapsids isolated from intact virions. These expression systems will definitely provide an alternative source for the production of the NP subunit or its assembled products in structural and functional analyses. To date, the nucleotide sequences of the NP gene of five NDV strains have been determined: AF2240, Beaudette C, La Sota, Ulster 2C, and Hitchner B1. A comparison of their predicted NP amino acid sequences revealed a high degree of identity ( 91 to 98% ) among the strains, but they share very low identity (