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Journal of Clinical Microbiology, February 2008, p. 750-753, Vol. 46, No. 2
0095-1137/08/$08.00+0     doi:10.1128/JCM.01587-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of Three New Virulent Newcastle Disease Virus Variants Isolated in China{triangledown}

Lei-Tao Tan,1 Huai-Ying Xu,1 You-Ling Wang,1 Zhuo-Ming Qin,1* Lei Sun,2 Wen-Jun Liu,2 and Zhi-Zhong Cui3

Institute of Poultry Science, Shandong Academy of Agricultural Sciences, Jinan 250100, China,1 Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China,2 College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, China3

Received 9 August 2007/ Returned for modification 16 October 2007/ Accepted 13 November 2007


   ABSTRACT
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Three cases of Newcastle disease virus (NDV) found in nature had the lentogenic motif 112G-R-Q-G-R-L117 in their fusion protein cleavage sites. However, both intracerebral pathogenicity and intravenous pathogenicity indexes showed that these NDV isolates were virulent. In comparison with the LaSota live virus vaccine, these viruses had significant genetic variations in the hemagglutinin-neuraminidase gene.


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Outbreaks of Newcastle disease (ND) frequently result in severe economic losses. It is caused by the ND virus (NDV) (2), an avian paramyxovirus that has been assigned to the new genus Avulavirus in the family Paramyxoviridae (13). The enveloped virus has a negative-sense single-stranded RNA genome of approximately 15 kb (5, 9). Among the six major proteins encoded by the NDV genome (11), the two interactive surface glycoproteins, the fusion (F) and the hemagglutinin-neuraminidase (HN) proteins, are involved in cell surface attachment and cell membrane fusion. Commonly, the F-protein cleavage site sequence is considered the primary molecular determinant of NDV virulence (8). However, recent work has demonstrated that NDV strains which carry exactly the same F-protein cleavage site had significant differences in their virulences. Furthermore, excluding the contribution of the F-protein cleavage site to virulence, the HN protein also contributed to virulence (6).

In China, a strict program of vaccination against ND during the past two decades resulted in a change in the occurrence of ND from pandemic to sporadic. Increasing poultry production and more selective immune pressure from hosts could enhance the evolutionary process of NDV (12). Outbreaks of ND have continuously been reported in poultry vaccinated with the lentogenic LaSota vaccine and flocks of wild birds (23). In this study, we characterized three new NDV isolates obtained from recent outbreaks in China in order to understand the potential relationship between these viruses and the LaSota vaccine.

Three field isolates were recently recovered from ND outbreaks in China and are described in Table 1. Filtrates from the processed tissues of the tracheas, oviducts, brains, and spleens of different hosts were inoculated into specific-pathogen-free eggs (Institute of Shandong Poultry Science), as reported previously (17, 18). The allantoic fluids were stored at –70°C after purification three times.


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  		TABLE 1. Pathogenicity and F-protein cleavage site sequences of NDV strains

 
The initial characterization of the isolates was performed by using the hemagglutination inhibition test with NDV-specific polyclonal antisera (1). The intracerebral pathogenicity index (ICPI) and the intravenous pathogenicity index (IVPI) were determined as described previously in the Office of International Epizootics manual of standards (24).

Viral RNA was extracted from the allantoic fluid by using the RNAgents total RNA isolation system (Promega, Madison, WI). The F- and HN-gene open reading frames from each of the three NDV isolates were amplified by reverse transcription-PCR (RT-PCR) (14, 22), which was performed by using a two-step RT-PCR program (37°C for 60 min; hold at 94°C for 3 min; and 32 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 2 min), the random primer 5'-ACGGG TAGAA-3', F-gene-specific primers (forward primer 5'-ATGGG CTCCA AACCT TCTAC-3' and reverse primer 5'-TTGTA GTGGC TCTCA TC-3'), and HN-gene-specific primers (forward primer 5'-TCCGT TCTAC CACAT CACCA-3' and reverse primer 5'-CGTCT TCCCA ACCAT CCTAT-3'). The amplicons of the F and HN genes were sequenced by Biotech (Shanghai, China). Nucleotide sequence editing, prediction of the amino acid sequences, and sequence analysis were performed by using the MegAlign program (Lasergene package; DNAStar, Madison, WI) (21). Phylogenetic trees were drawn by using the neighbor-joining method of the MEGA program (version 3.1), based on the variable-region (nucleotides [nt] 47 to 420) nucleotide sequences of the F gene and the deduced amino acid sequences of the HN gene (10).

The F protein, synthesized as the nonfunctional precursor F0, must be cleaved into disulfide-linked F1 and F2 polypeptides by host proteases to become fusogenic and is an important determinant of the pathogenicity of NDV (15, 16, 19). Previous studies comparing the precursor peptide F amino acid sequences of NDV strains that varied in their virulences showed that virulent viruses had the amino acid sequence 112R/K-R-Q-K/R-R116 at the C terminus of the F2 protein and phenylalanine at residue 117 at the N terminus of the F1 protein. However, viruses with low levels of virulence had the sequence 112G/E-K/R-Q-G/E-R116 at the C terminus of the F2 protein and leucine at residue 117 at the N terminus of the F1 protein (4). On the basis of the sequence of the variable region (nt 47 to 420) of the F gene (12), isolates SQZ/04, JS05/03, and QE01/99 from China appeared to form a separate cluster with genotype II strains, including strains LaSota, TEX/48, and B1 (Fig. 1). The amino acid sequences of the F-protein cleavage site showed that the three field isolates had the common motif 112G-R-Q-G-R-L117, consistent with the sequence of lentogenic vaccine viruses LaSota and B1. These field isolates would be classified as lentogenic viruses on the basis of the F-protein cleavage site motif. Interestingly, the biological tests suggested that strains SQZ/04, JS05/03, and QE01/99 were virulent strains, with ICPI values of 2.00, 1.75, and 1.81, respectively, and IVPI values of 2.68, 2.58, and 2.24, respectively (Table 1). It was the specific case in which highly virulent viruses were shown to emerge from lentogenic live virus vaccines. As reported experimentally, some avirulent viruses have the potential to become highly virulent in birds (25). Although the reason is unknown, these velogenic NDV isolates could have originated in nature from lentogenic vaccine viruses. These results also show that evaluation of the virulence of NDV by the characteristic pattern of amino acid residues at the F-protein cleavage site is not efficient.


Figure 1
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  		FIG. 1. Phylogenetic relationships between the three NDV isolates (in boldface) and reference viruses with different genotypes based on the variable-region nucleotide sequences (nt 47 to 420) of the F gene. The phylogenetic tree was generated by the neighbor-joining method with the MEGA program (version 3.1).

 
The F-gene full-length amino acid sequences of the three field isolates were compared with those of reference viruses representing different genotypes. The results showed that they shared 99.3 to 99.6% identities with the LaSota vaccine virus, which was greater than the 96.9 to 99.1% identities with viruses TEX/48 and B1, the 92.4 to 92.8% identities with genotype I NDV (Queensland/V4), the 91.3 to 91.7% identities with genotype III NDV (Australia-Victoria), the 91.5 to 92.0% identities with genotype IV NDV (Herts/33), the 87.9 to 88.2% identities with genotype V NDV [Turkey/USA(ND)/43084/92], the 89.9 to 90.2% identities with genotype VI NDV (Chicken/Kenya/139/90), the 88.4 to 88.8% identities with genotype VII NDV (SGM/01), and the 88.8 to 89.3% identities with genotype VIII NDV (AF2240). Furthermore, among the LaSota, SQZ/04, JS05/03, and QE01/99 viruses, viral cysteine residues for disulfide bonds and potential glycosylation sites (3) were conserved in all viruses with the exception of virus SQZ/04, which had an R-for-C substitution at amino acid position 374. These results suggest that the three velogenic viruses might be generated from the LaSota virus, since it was widely used as a live virus vaccine.

The increase in virulence of NDV strains is not caused by another mutation in the F gene but is probably caused by a mutation(s) elsewhere in the NDV genome (7). High degrees of similarity of the F-gene cleavage sites and significant differences in virulence between the LaSota virus and these three field isolates were found in this study, which implied that additional factors might contribute to the virulence of NDV.

Data from recent publications indicate that the effect of the HN protein on virulence was prominent and that both the stem region and the globular head of the HN protein seem to be involved in the determination of virulence (6). The glycoprotein plays an important role in both recognizing sialic acid-containing receptors on cell surfaces and promoting the fusion activity of the F protein (20). This implies that the immune system of the hosts exerts pressure on the HN protein. In the comparison of the HN full-length amino acid sequences, the SQZ/04, JS05/03, and QE01/99 isolates showed only 87.0 to 88.8% identities with vaccine virus strain LaSota, which was less than the 95.6 to 99.8% identities with the five prevailing genotype VII strains SSX/03, SKY/03, JS04/04, SGM/01, and JS01/01, which have a common 112R-R-Q-K-R-F117 motif with a highly virulent F gene (Table 1). In addition, the phylogenetic relationships based on the HN amino acid sequence showed that the three field NDV isolates appeared to form a cluster with the viruses isolated from 1972 to 2004, whereas strains isolated from 1932 to 1967 formed a different cluster (Fig. 2). By comparison, the three field isolates and the five prevailing reference viruses all lost a potential glycosylation site at residues 538 to 540 of the HN gene that existed in the LaSota virus. These viruses had a C residue at position 123 of the HN gene that was not present in the LaSota virus; a similar result was also reported previously (3). These results demonstrate that there have been distinct mutations in the HN gene among recent NDV strains, and the frequency of mutation is closely related to the times of outbreaks. This might explain the cause for the differences in viral virulence and the frequent outbreaks of ND in immunized bird flocks, especially outbreaks in China during the past several years.


Figure 2
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  		FIG. 2. Phylogenetic relationships between the three NDV isolates (in boldface) and reference viruses based on the deduced amino acid sequences (residues 1 to 571) of the HN gene. The phylogenetic tree was generated by the neighbor-joining method with the MEGA program (version 3.1).

 
In this study, we found that virulent NDVs that possess the lentogenic motif 112G-R-Q-G-R-L117 at the F-protein cleavage site were already in existence. Furthermore, isolates SQZ/04, JS05/03, and QE01/99 might be generated in nature from the LaSota live vaccine virus strain under host immune pressure, and the HN protein can contribute to viral virulence; thus, the F-gene cleavage site is not the only factor determining NDV virulence.


   ACKNOWLEDGMENTS
 
This work was supported by the Shandong Provincial Program for Key Science and Technology Projects (grant 030317), the Shandong Provincial Natural Science Foundation (grant 031020101), and the Innovation Fund from the Shandong Academy of Agricultural Sciences (grants 2006YCX024 and 2007YCX017).


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Poultry Science, Shandong Academy of Agricultural Sciences, 58, Northern Road of Huangtai, Jinan 250100, China. Phone and fax: 86-531-88961344. E-mail: qinzm1997{at}163.com Back

{triangledown} Published ahead of print on 5 December 2007. Back


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Journal of Clinical Microbiology, February 2008, p. 750-753, Vol. 46, No. 2
0095-1137/08/$08.00+0     doi:10.1128/JCM.01587-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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