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Department of Molecular Genetics and Microbiology, Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
ABSTRACT
Newcastle disease virus (NDV) strains are classified as having high (velogenic), intermediate (mesogenic), or low (lentogenic) pathogenesis and virulence in chickens. Recent studies have established that the hemagglutinin-neuraminidase (HN) protein plays an important role in viral tropism and virulence. A monoclonal antibody (AVS-I) has previously been shown to be specific for lentogenic strains of NDV (Srinivasappa et al., Avian Dis. 30:562-567, 1986) and is routinely used to identify these strains. We have used competition antibody binding assays with a previously characterized panel of monoclonal antibodies, binding to chimeric HN proteins, and the characterization of an escape mutant to localize the binding site of AVS-I to the extreme carboxy terminus of the protein. In addition, we have shown that AVS-I does recognize at least one mesogenic strain and one velogenic strain of the virus, calling into question the potential of this antibody as a diagnostic reagent for avirulent NDV strains.
TEXT
Newcastle disease virus (NDV) is a member of the genus Avulavirus of the Paramyxoviridae family of enveloped negative-stranded RNA viruses. The envelope of NDV virions contains two types of glycoprotein spikes, the hemagglutinin-neuraminidase (HN) and fusion (F) proteins. HN is responsible for attachment of the virus to sialic acid-containing cell surface receptors. It also possesses neuraminidase (NA) activity that cleaves sialic acid from progeny virus particles to prevent viral self-aggregation. HN also promotes the fusion activity of the F protein responsible for virus-cell and cell-cell fusion (18).
NDV causes respiratory, neurological, or enteric disease in many species of birds, resulting in significant losses to the poultry industry worldwide. Strains of the virus are classified into three pathotypes based on the severity of disease in chickens. Avirulent strains that produce mild or asymptomatic infections are termed lentogenic, whereas virulent strains that cause acute infections with high mortality are termed velogenic. Strains of intermediate virulence are termed mesogenic (1). Velogenic strains are further categorized as either neurotropic or viscerotropic.
It is widely accepted that cleavage of the fusion protein precursor (F0) is the primary determinant of NDV virulence. F0 is cleaved at a basic amino acid-rich region, resulting in the formation of the active fusion protein consisting of disulfide-linked F1 and F2 polypeptides (18). Virulent strains have four basic residues in the cleavage site, whereas avirulent strains have only two (3, 20). The F0 of virulent NDV strains is cleaved by host proteases found in a wide range of tissues, whereas that of avirulent strains is cleaved only by trypsin-like proteases secreted by a limited number of tissues in the respiratory and intestinal tracts (14).
However, the susceptibility to cleavage of the F protein is not the sole determinant of NDV virulence. Modification of a lentogenic F cleavage site to a velogenic one increased virulence, but not to the level of velogenic strains (15, 16). This indicates that other viral proteins in addition to F also contribute to virulence. Huang et al. (4) recently showed that the HN protein plays a role in viral tropism and virulence. The HN gene of the Beaudette C (BC) mesogenic recombinant strain rBeaudette C was exchanged with that of lentogenic recombinant strain rLaSota, creating a BC virus having the HN of LaSota and a LaSota virus having the HN of BC. Pathogenicity studies showed that the BC virus having the HN of LaSota decreased in virulence and the LaSota virus having the HN of BC increased in virulence, indicating that HN plays a role in this process.
We previously characterized a panel of monoclonal antibodies (MAbs) raised against the HN glycoprotein of the velogenic Australia-Victoria/32 (AV) strain of NDV. These MAbs were used in competition antibody binding assays and additive neutralization assays to delineate seven antigenic sites that form a continuum on HN (5, 6, 10). Escape mutants were selected with MAbs to each site and sequenced to identify the following epitopes: site 1 (residue 345), site 2 (residues 513, 514, 521, and 569), site 3 (residues 263, 287, and 321), site 4 (residues 332, 333, and 356), site 12 (residues 494 and 516), site 14 (residues 347, 350, and 353), and site 23 (residues 193, 194, and 201) (13). Only site 14 MAbs recognize a linear epitope, defined by residues 341 to 355; all other sites are conformational (13). In addition, antibodies to sites 1, 4, and 14 recognize a broad range of strains, while those to the other sites exhibit various degrees of strain specificity (5, 12).
Srinivasappa et al. (19) previously isolated a monoclonal antibody (AVS-I), raised against the avirulent LaSota strain of NDV, which reacted exclusively in hemagglutination inhibition (HI) assays with lentogenic strains of NDV (B1-Hitchner, LaSota, Queensland V4, and Ulster), though it did not react with two such strains (ENG F and NEB GOL). AVS-I also exhibited HI activity towards 10 commonly employed commercial B1-Hitchner and LaSota vaccine strains. Most importantly, AVS-I did not react with three mesogenic (ENG P3R10, Roakin, and Kimber) or six velogenic (GB Texas, Largo, Calif 1083, KM, P1307, and P5658) strains. These data suggested that this antibody recognizes an epitope that is conserved in lentogenic strains and have led to its use in identifying strains of NDV belonging to this pathotype. These findings also raise the possibility that the AVS-I epitope may colocalize with a determinant of virulence in HN.
To further characterize antibody AVS-I and the epitope it recognizes, we have (i) determined its specificity for several additional strains of the virus, (ii) mapped its binding to HN in competition with our own antibodies, (iii) determined its functional inhibition profile, and (iv) isolated and sequenced an AVS-I escape mutant. We have shown that AVS-I binds to a conformational epitope at the carboxy terminus of the protein that overlaps with our original sites 1 and 2 and only weakly inhibits the NA activity of the protein. In addition, we have shown that the antibody, which was previously thought to be specific for avirulent strains of NDV, actually recognizes individual mesogenic and velogenic strains. The relevance of these findings to virulence and the suitability of AVS-I as a diagnostic reagent is discussed.
To test the avirulent specificity of AVS-I, an enzyme-linked immunosorbent assay (ELISA) was performed using intact virions of several additional strains of the virus not tested previously with this MAb. These include lentogenic, mesogenic, and velogenic strains. The two lentogenic strains are B1-Hitchner/48 (B1) and Ulster/64 (U), which have F cleavage sites with a low basic residue content. The mesogenic strains include NJ-Roakin/FRB/46 (F) and Massachusetts-4F/46 (M). The velogenic strains include Australia-Victoria/32 (AV), Texas-GB/48 (GB), Iowa-Salsbury/49 (IS), Kansas-Leavenworth/48 (L), and California-RO/44 (RO). All mesogenic and velogenic strains have a high basic residue content in the F cleavage site, which is evidence alone that other determinants besides the cleavage site, possibly on HN, contribute to virulence.
Each purified virus stock was diluted to 20 μg/ml with phosphate-buffered saline (PBS), and 50 μl of this solution were placed into each well of a 96-well microtiter plate. The wells were allowed to dry overnight and blocked with 2% agamma horse serum. Fifty microliters of a 1:2,700 dilution of AVS-I-containing ascites fluid were added to each well and incubated at 37°C for 1 h. Horseradish peroxidase-conjugated goat anti-mouse antibody (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added at a 1:1,000 dilution and incubated at 37°C for 1 h. Following the addition of SureBlue TMB microwell peroxidase substrate (Kirkegaard and Perry Laboratories), the absorbance was measured at 650 nm. Antibody to site 1 was used as a positive control. This antibody has previously been shown to have broad specificity for a variety of NDV strains. Antibody to site 23, previously shown to be highly specific for the AV strain, was used as a negative control. Consistent with the results of the original study (19), AVS-I recognizes the lentogenic strains B1 and U and does not recognize mesogenic strain F or velogenic strains GB, IS, L, and AV (Table 1). However, most unexpectedly, AVS-I does recognize mesogenic strain M and velogenic strain RO. Hence, these strains are exceptions to the specificity of AVS-I for avirulent strains. This finding calls into question the suitability of AVS-I as a marker for avirulent strains of the virus.
The ability of AVS-I to inhibit the hemagglutinating (HA) activity of our panel of NDV strains was also determined as described previously (7) with some modifications. Serial twofold dilutions of ascites fluid (25 μl) were incubated with four HA units of purified virus in 25 μl PBS with Ca2+ and Mg2+ at 37°C for 30 min. After adding chicken erythrocytes, plates were incubated at 4°C for 1 h. An antibody to site 1 was used as a positive control, while antibody to site 4 served as a negative control. As shown in Table 1, AVS-I inhibits the HA activity of avirulent strains B1 and U but not that of strain F or AV and only marginally that of GB, IS, or L, consistent with the ELISA results. AVS-I recognizes the virulent strain RO only at a low level, whereas it had bound to this strain quite efficiently in the ELISA. However, AVS-I does inhibit the HA activity of strain M at a very high titer.
Next, we determined the ability of AVS-I to neutralize the panel of NDV strains. For this assay, 107 PFU/ml of each virus was incubated at 25°C for 1 h with an equal volume of AVS-I ascites fluid diluted to an antibody concentration of 50 μg/ml in Hanks' balanced salt solution (Invitrogen Life Technologies, Carlsbad, CA). Aliquots of this mixture were plated on chicken embryo cells and plaque assays were performed as described previously (8). For avirulent strains that require trypsin to form plaques, cells were overlaid with medium 199 (Invitrogen) supplemented with 0.1% NaHCO3, 2.5% tryptose phosphate broth, 0.9% agar, and 10 μg/ml trypsin. Treatment with rabbit anti-mouse immunoglobulin (RAM Ig) (Litton Bionetics, Kensington, MD) was carried out to reduce the persistent fraction of nonneutralized virus (8).
Table 1 shows that there are three categories of neutralization by AVS-I. Members of the first group are neutralized to a significant extent by AVS-I alone. This group includes strains B1 and RO, treatment of which with AVS-I results in persistent fractions of 5 to 10%. Addition of RAM Ig results in complete neutralization of both strains. The addition of a second antibody specific for a nonneutralizing first antibody has long been known to result in an enhanced level of neutralization. This phenomenon has previously been demonstrated with antibodies to NDV (6) and to other viruses (reviewed in reference 6).
Members of the second group, which includes strains AV, F, L (Table 1), and Eng F (data not shown), are not neutralized by AVS-I, either alone or with RAM Ig, suggesting that the antibody does not bind at all to these strains. These results are consistent with the ELISA data in Table 1 and the original HI data for Eng F (19).
Members of the third group exhibit an apparently paradoxical neutralization profile. This group, which includes strains U and M (Table 1), as well as LaSota (data not shown), are not neutralized by AVS-I alone but are neutralized upon the addition of RAM Ig. We have previously characterized NDV-AV mutants of this type selected with antibodies to HN sites 1, 2, and 4 (8). A likely explanation for this is that AVS-I binds to these strains but the binding is not of sufficient avidity to result in neutralization. The RAM Ig second antibody stabilizes its binding, possibly by binding bivalently to two AVS-I molecules. Consistent with this, binding of AVS-I to both strain U and strain M can be detected by ELISA (Table 1).
One caveat that emerges from these studies is that HI activity does not necessarily correlate with neutralizing activity. For example, AVS-I exhibits an extremely high HI titer for strain M but is unable to neutralize it without the addition of RAM Ig, while it can neutralize strain RO despite only weak HI activity. This suggests that the mechanism of neutralization by antibody AVS-I may not involve preventing virus attachment. We have previously characterized antibodies to two sites on HN that neutralize infectivity and block fusion, despite the lack of HI activity (11).
To map the binding site of AVS-I on HN relative to our panel of MAbs, reciprocal competition antibody binding assays were performed using B1 virus. By necessity, competing MAbs in this experiment are restricted to those members of our panel that recognize this strain. These include antibodies to sites 1, 14, 4, and 2 but not those to sites 12, 23, and 3 (5, 12; data not shown). All antibodies were adjusted to an initial concentration of 2.5 mg/ml. In the first set of experiments, microtiter plates were coated with 1 μg/well of B1 virus and blocked with 2% agamma horse serum. A serial dilution of the first antibody (unlabeled AVS-I) was added and the plate was incubated at 37°C for 1 h. After several washes with PBS, the second antibody (biotinylated AVS-I or biotinylated antibody to either site 1, 14, 4, or 2) was added and again incubated at 37°C for 1 h. Finally, streptavidin-horseradish peroxidase (Zymed Laboratories Inc., South San Francisco, CA) was added at a 1:5,000 dilution for 1 h at 37°C and absorbance measured as described before. As shown in Fig. 1A, AVS-I blocks itself and antibodies to sites 1 and 2 at the same concentrations but does not block antibodies to either site 14 or site 4.
Figure 1B shows the results of the reciprocal experiment in which the second antibody is biotinylated AVS-I. Again, AVS-I blocks itself and the antibody to site 2 also blocks AVS-I. The antibody to site 1 blocks AVS-I, but only at higher antibody concentrations. The antibody to sites 14 and 4 does not block AVS-I. Thus, AVS-I and the antibody to site 2 compete with each other reciprocally. AVS-I efficiently blocks the antibody to site 1, but the antibody to site 1 blocks AVS-I only at very high concentrations. These data suggest that the binding site of AVS-I is located closest to antigenic site 2 and also partially overlaps with site 1. One possibility that could account for the nonreciprocal competition between AVS-I and the site 1 antibody is that the binding of AVS-I may induce a nonreciprocal conformational change in the protein that alters site 1.
To further characterize the functional inhibition profile of MAb AVS-I, its ability to inhibit the NA activity of avirulent strain B1 was determined. Virus was diluted to 0.35 mg/ml in 0.1 M sodium acetate (pH 6.0). Forty microliters of this solution was incubated at 37°C for 1 h with an equal volume of 0.2 mg/ml affinity-purified AVS-I antibody. Twenty microliters of this mixture was incubated with 0.5 ml of 625 μg/ml of neuraminlactose (Sigma Chemical Co., St. Louis, Mo.) at 37°C for 1 h. The amount of N-acetyl-neuraminic acid released was determined as described previously (7). The antibody to site 2 was used as a positive control. This antibody has previously been shown to inhibit the NA activity of the AV strain. An MAb specific for the nucleocapsid protein (NP) was used as a negative control. For the avirulent strain B1, the percentages of NA activity remaining are 97% ± 7%, 25% ± 3%, and 68% ± 10% for the NP, site 2, and AVS-I antibodies, respectively. This indicates that AVS-I partially inhibits the NA activity of strain B1. To be certain that the effect is not dose dependent, a threefold higher concentration of each antibody was also tested. The percentages of NA activity remaining were not significantly different from those obtained with the lower antibody concentration, i.e., 102% ± 5%, 23% ± 3%, and 72% ± 8% for the NP, site 2, and AVS-I antibodies, respectively. As a control, the AVS-I antibody does not inhibit the NA activity of the virulent strain AV (106% ± 26% NA activity remaining), consistent with its inability to recognize this strain. The results with B1 suggest that, although the AVS-I epitope maps close to the epitope recognized by the site 2 MAb, it maps further from the NA active site than site 2.
To determine whether the epitope recognized by AVS-I is linear or conformational, a Western blot was performed. Polyacrylamide gel electrophoresis was carried out under reducing conditions using 20 μg of viral proteins from either the B1 or U strain. The proteins were transferred to a nitrocellulose membrane and probed with MAbs to site 14 or 4 or AVS-I. Antibody to site 14 recognizes a conserved, linear epitope, while antibody to site 4 recognizes a conserved, conformation-dependent epitope (5, 12, 13). As expected, the site 14 antibody, but not the site 4 antibody, binds to HN in the Western blot. AVS-I does not bind to HN, which suggests that it recognizes a conformation-dependent epitope (data not shown).
To begin to map the epitope recognized by AVS-I, HN chimeras consisting of domains from the virulent strain AV and the avirulent strain B1 were constructed by taking advantage of a unique NspI restriction site. This made it possible to exchange amino acids 344 through 571 of AV HN with amino acids 344 through 577 of B1 HN, creating the HN chimeras AV-B1 and B1-AV (Fig. 2). These chimeras were expressed in BHK-21 cells (American Type Culture Collection, Manassas, VA) as described previously (9). To determine whether the chimeras are functional, a hemadsorption (HAd) assay was performed (9). Each of the HN chimeras hemadsorbs at a level comparable to that of wild-type (wt) HN, indicating that they are expressed and are functional. To assess binding of AVS-I to the HN chimeras, flow cytometric analysis was performed. A cocktail of antibodies to sites 1, 14, and 2 was used to confirm that the chimeras are indeed expressed at the cell surface. Binding is expressed as a percentage of that of wild-type B1 HN. As shown in Fig. 2, AVS-I binds to B1 HN and the AV-B1 chimera, whereas it does not bind to either AV HN or the B1-AV chimera. This suggests that AVS-I binds to an epitope in the C-terminal region of B1 HN between amino acids 344 and 577.
To begin to identify the HN amino acid residues in the epitope recognized by AVS-I, an escape mutant virus was isolated. B1 virus was passaged three times in eggs. The allantoic fluid was treated with AVS-I and plated on chicken embryo cells in the presence of trypsin. A plaque assay was performed as described previously (8). Treatment with AVS-I resulted in the usual persistent fraction of nonneutralized virus. The neutralization studies with this strain (Table 1) suggest that, despite its escape from neutralization, this persistent fraction does have antibody bound to it. Thus, to make variant selection possible, AVS-I-neutralized virus was treated with RAM Ig. This reduces the persistent fraction to 0.1% and makes possible the isolation of escape mutants. One escape mutant, AVS-B1, was isolated and plaque purified. Genomic RNA sequencing of the HN gene of escape mutant AVS-B1 revealed the presence of a G570R mutation. The presence of this mutation, and only this mutation, in AVS-B1 HN was confirmed by reverse transcription-PCR.
The ability of antibody AVS-I to recognize AVS-B1 virus was determined by ELISA. The AVS-B1 virus is not recognized by AVS-I, consistent with its ability to escape neutralization by the antibody (data not shown). Antibodies to sites 1 and 14 serve as positive controls, and antibody to site 23 serves as a negative control.
To further establish that residue G570 is important for the binding of AVS-I, the G570R mutation was introduced into B1 HN through site-directed mutagenesis as described previously (9) using the mutagenic primer 1696-CTCAAAGATGATAGGGTTCGCGAGGCCAGGTC-1727 with the mutated codon in bold. Screening for the mutant was facilitated by the introduction of an NruI site (underlined). The G570R-mutated HN was expressed in BHK cells and HAd inhibition and flow cytometric analyses were performed to assess the ability of AVS-I to recognize it. The former was performed as described previously (9) using 300 μl of AVS-I ascites fluid with the amount of HAd quantitated by lysing of the adsorbed guinea pig erythrocytes in ammonium chloride and measurement of absorbance at 540 nm. Antibody AVS-I did not inhibit the HAd activity of G570R-mutated B1-HN, resulting in an absorbance of 104% of that obtained with untreated monolayers. As controls, AVS-I completely blocked the HAd activity of wt B1-HN and antibody to site 14 completely inhibited that of both the wt and mutated proteins.
For flow cytometry, a cocktail of antibodies to sites 1, 14, and 2 was used to confirm that the G570R-mutated HN is expressed at a level comparable to that of wild-type HN. Unlike the HAd inhibition results, we are able to detect an interaction between AVS-I and G570R-mutated B1-HN, although significantly decreased compared to the wt protein (39 ± 3%). This finding, along with our inability to identify another mutation in the mutant virus, suggests that the G570R mutation does not completely eliminate the ability of AVS-I to recognize the virus but does reduce the avidity of the interaction to a level insufficient to block infectivity or attachment to receptors on red blood cells.
The results of our functional inhibition studies with AVS-I are reminiscent of previous data obtained with antibody to site 2 (12). Even though the antibody to this site was made against the virulent strain AV, it has the same strain preference as AVS-I. The antibody to site 2 is better able to neutralize avirulent strains than virulent ones: the persistent fractions of avirulent strains B1, LaSota, and W are lower than those of virulent strains Italy-Milano/45, Israel-HP/53, L, and F. A distinct exception to this is the virulent strain RO, which is neutralized to a threefold-greater extent than AV. Hence, the strain specificity of AVS-I is similar to that of antibody to site 2. Also, this represents another example of the HN of virulent strain RO being more similar to the HN proteins of avirulent strains. It also points to a relationship between the C-terminal end of HN and virulence.
Given all of this, it would not be surprising for residue 570 to be part of a determinant of virulence in HN. Consistent with this possibility is the fact that it is situated close to the sialic acid binding site (9). Furthermore, a cold-adapted temperature-sensitive B1 virus, which does not bind AVS-I (2), has the identical G570R mutation (Bruce Seal, personal communication). However, the escape mutant, AVS-B1, does not exhibit an altered mean embryo death time relative to the wt virus, suggesting that the mutation does not affect virulence (data not shown). There is also not a consistent relationship between the nature of residue 570 and virulence. Though virulent strains Chiba/85 and Ibaragi/85 have an arginine at position 570, most virulent strains, including Beaudette C/45, Texas GB/48, Australia-Victoria/32, Herts/33, and Italien/45, have a glycine at this position (17). Thus, despite being part of the AVS-I epitope, residue G570 appears not to be a major determinant of virulence. However, given the complexity of antibody-binding epitopes, other residues that make up the AVS-I epitope could certainly contribute to virulence. A complete understanding of the relationship between AVS-I recognition of a virus and virulence will require a complete fine mapping of each of the amino acids that contributes to the epitope.
ACKNOWLEDGMENTS
We gratefully acknowledge Elizabeth Corey, Paul Mahon, Vanessa Melanson, and Anne Mirza for critical reading of the manuscript. We also acknowledge the gift of hybridoma AVS-I from David Snyder, as well as thank Kemal Karaca for the B1 HN clone and Trudy Morrison for the AV HN clone.
This work was made possible by grant AI-49268 from the National Institutes of Health.
Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.
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