- Email: [email protected]
Genotypic and pathotypic characterization of Newcastle disease virus isolated from racing pigeons in China
IMMUNOLOGY, HEALTH, AND DISEASE Genotypic and pathotypic characterization of Newcastle disease virus isolated from racing pigeons in China Mengda Liu,∗ Yajin Qu,† Fangkun Wang,† Sidang Liu,† and Honglei Sun∗,1 ∗
Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China; and † College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian 271018, China fusion (F) protein confirmed that the isolate contained the virulent motif 112 RRQKRF117 at the cleavage site, but it caused no appearance disease in chickens and ducks. However, the isolate had virulence in pigeons, resulting in severe nervous signs and highly mortality. Pigeons were considered as a potential source of NDV infection and disease for commercial poultry flocks. Therefore, new vaccines to prevent the NDV infection in the pigeon flocks should be developed as soon as possible, and strict biosecurity measures should be taken to reduce the risk of pigeon Newcastle disease outbreaks.
Key words: Newcastle disease virus, racing pigeon, phylogenetic analysis, pathology 2015 Poultry Science 94:1476–1482 http://dx.doi.org/10.3382/ps/pev106
INTRODUCTION Newcastle disease is a highly contagious viral disease of poultry and other bird species caused by the Newcastle disease virus (NDV), a member of the genus Avulavirus in the family Paramyxoviridae (Mayo, 2002; Nagai et al., 1976). The genome of NDV is a nonsegmented, single-stranded, negative-sense RNA molecule of approximately 15,186 nucleotides (nt) that contains 6 genes encoding the 6 structural proteins (from the 3 to 5 terminus): nucleoprotein, phosphoprotein, matrix, fusion (F), hemagglutinin-neuraminidase (HN), and the large protein (Chambers et al., 1986). NDV strains can be classified as high virulence (velogenic), intermediate (mesogenic), or low virulence (lentogenic) based on various biological parameters, such as the mean death time (MDT) of chicken embryos infected with the minimum lethal dose of virus, the intracerebral pathogenicity index (ICPI) in 1-day-old chicks, and the intravenous pathogenicity index (IVPI) in 6week-old chickens. The primary molecular determinant C 2015 Poultry Science Association Inc. Received December 4, 2014. Accepted February 27, 2015. 1 Corresponding author: [email protected]
for NDV pathogenicity is the amino acids of the F protein cleavage site (Nagai et al., 1976; Ogasawara et al., 1992). NDV has a wide host range with most orders of birds that reported to have been infected by the virus. More commonly affected species include chickens, turkeys, pigeons, and ducks (King, 1996; Lana et al., 1988; Zhang et al., 2011). Variant strains of NDV are associated with infections of pigeons, known as pigeon paramyxovirus Type 1 (PPMV-1), which can be characterized by unique monoclonal-antibody-binding profiles (King, 1996; Lana et al., 1988). After its first emergence in the Middle East (late 1970s) (Lister et al., 1986), PPMV-1 spread worldwide (Alexander, 1985; Lister et al., 1986; Vindevogel and Duchatel, 1988), and several Newcastle disease outbreaks in chickens have been attributed to PPMV-1 (Alexander et al., 1984). Therefore PPMV-1 presents which makes it a real threat to the poultry industry. The frequent isolation of PPMV-1 from pigeons across China necessitates an assessment of the risk potential of these viruses causing disease in poultry (Liu et al., 2006; Yu et al., 2001). In the present study, a PPMV-1 isolate from outbreaks in racing pigeons in China was pathotypically and genotypically characterized.
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ABSTRACT A Newcastle disease virus (NDV) isolated from an outbreak in racing pigeons in China was characterized in this study. Complete gene of the NDV isolate was sequenced and phylogenetic analysis. Pathogenicity experiment was carried out in pigeons, chickens, and ducks. Phylogenetic analysis revealed that the strain clustered with the Class II viruses, has highly phylogenetically similar to NDV strains isolated from pigeons in China, but was distant from the viruses prevalence in chickens and vaccine strains used in China. The deduced amino acid sequence of the cleavage site of the
CHARACTERIZATION OF NEWCASTLE DISEASE VIRUS ISOLATED FROM PIGEONS
MATERIALS AND METHODS Virus Isolation and Identification
isolate, virus-infective allantoic fluid was diluted 10fold in saline from 10−1 to 10−8 . For each dilution, 3 eggs were used to inoculate 0.1 mL diluted virus via the allantoic cavity. After 72 h, virus infection was assessed by HA assay of egg allantoic fluid. EID50 was calculated using the method of Reed and Muench (1938).
Phylogenetic Analysis and Molecular Characterization Viral RNA was extracted using RNeasy Mini Kit (Qiagen, Chatsworth, CA). reverse transcription and PCR was performed under standard conditions as previously described (Liu et al., 2006). Primer sequences used to generate for the overlapping subgenomic cDNA fragments and for genome sequencing are available upon request. PCR products were purified using the QIA quick PCR Purification Kit (Qiagen, Valencia, CA), and then sequenced by the Beijing BGI-GBI Biotech Co., Ltd. Reference NDV genomic sequences were obtained from GenBank, and these included current vaccine strains, typical prevailing isolates in China, and the reference strains for each known NDV genotype. These NDV sequences and the complete coding sequences of the NDV isolate were aligned and analyzed using the ClustalW multiple alignment algorithm in the MegAlign program of the DNASTAR software suite (version 3.1; DNAstar, Madison, WI). Phylogenetic analysis was performed using software MEGA 4.1. (DNAStar Inc.) by the distance-based neighbourjoining method (1,000 replicates for bootstrap).
Animals To investigate the biological characteristics and pathogenicity of the NDV isolate, SPF chickens, NDV antibody-negative pigeons, and NDV antibody-negative Beijing ducks were used. All animal research was approved by Beijing Administration Committee of Laboratory Animals under the leadership of the Beijing Association for Science and Technology; the approve identifier is SYXK (Beijing) 2007–0023.
Assessment of the Biological Characteristics of the Isolate The pathogenic potential for the isolated viruses was evaluated using standard assay methods to determine the MDT in 10-day-old chick embryos, the ICPI in 1-day-old chickens, and IVPI in 6-weeks-old chickens. For MDT test, virus-infective allantoic fluid was diluted 10-fold in saline from 10−6 to 10−10 . For each dilution, 5 eggs were used to inoculate 0.2 mL diluted virus via the allantoic cavity. After 24 h, the eggs were examined for mortality at 2-h intervals for 120 h. The MDT was the mean time in hours for the lowest dilution to kill all eggs of that dilution. To test the 50% embryo infectious doses (EID50 ) of the
Animal Experiments Twenty 1-month-old NDV antibody-negative pigeons, twenty 6-weeks-old SPF chickens, and twenty 2weeks-old NDV antibody-negative Peking ducks were randomly divided into 2 groups, respectively. The challenge procedure was birds were inoculated via the intranasal route with 106 EID50 of the virus in a 0.2 mL volume or 0.2 mL PBS as a mock-infected control. All birds were clinically monitored every day for signs of disease (e.g., disheveled feathers, lethargy, fever or paralysis) and mortality. Dead birds were immediately necropsied for the determination of gross lesions, and their tissue samples (trachea, lung, brain, spleen, small intestine, proventriculus, and kidney) were collected and fixed in 10% neutral buffered formalin for histological observation. In addition, antigens of NDV present in the tissue of virus inoculation birds were further examined using immunohistochemistry, which employed an HN-protein-specific mouse monoclonal antibody. Serum samples were collected from all birds before inoculation and at 14 d postinfection (dpi) for NDV-specific antibody detection using an HI test with 1% chicken red blood cells.
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In February 2012, a flock of racing pigeons displayed severe nervous signs in Shandong Province of China. Tracheal and cloacal swabs were collected from the diseased pigeons. Swabs samples were maintained in transport medium containing antibiotics and kept at 4◦ C during transportation to the laboratory. Samples were centrifuged for 10 min at 3,000 × g at 4◦ C and 0.2 mL supernatant was used to inoculate the allantoic cavities of 9- to 11-day-old specific pathogen-free (SPF) embryonated chicken eggs, and incubated at 35◦ C for 72 h. Allantoic fluids were then harvested and tested for haemagglutinin (HA) activity. Positive isolates were characterized by HA inhibition (HI) tests using NDV, H5, H9 subtype avian influenza virus, and egg drop syndrome virus antibodies. The pigeon virus showed HA activity, which was inhibited by NDV antibody, but revealed no cross-reaction to avian influenza virus and egg drop syndrome virus antibodies in HI test. The strain was designated Pigeon/China/SD069/2012. To purify the virus, 10-fold dilutions of a virus stock are prepared, and 0.1-mL aliquots are inoculated onto baby hamster kidney cell monolayers. After an incubation period, the monolayers are covered with a nutrient agar. After 48 h, each infectious particle produces a plaque. The virus was purified 3 times using a plaque technique before being propagated in the allantoic cavities of 10-dayold SPF embryonated chicken eggs. Virus stocks were stored at −80◦ C until use.
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RESULTS Biological Characteristic Assessment of the SD069 Isolate Initial biological characterization of the isolate strain SD069 included MDT, ICPI, and IVPI were determinations. The virus exhibited MDT of 96 h and the ICPI values were 0.6 for the moderately virulent isolate. The virus did not produce obvious signs or lesions in all the 10 chickens within 10-d obvious period, indicating the IVPI was 0.
Sequence and Phylogenetic Analysis The nucleotide sequence data were deposited into the Genbank database and the accession numbers was
KP861633. The total length of the strain was 15,192 nt. Compared with the NDV Lasota strain, the isolated strain bears a 6-nt insertion (TCCCCA) in positions 1,647 to 1,648 nt of the nucleoprotein gene. Proteolytic cleavage site motifs (Residues 112 to 117) for the F0 protein in the isolate were analyzed. The strain has a virulent motif (112 RRQKRF117 ) composed of multibasic amino acids at the precursor F (F0) cleavage site. This motif is commonly found in strains that are highly virulent in chickens (Collins et al., 1993; Rui et al., 2010). Phylogenetic analysis was conducted based on complete nucleotide sequences. Nucleotide sequence data of 32 NDV reference strains obtained from the GenBank database were used for comparison. As shown in Figure 1, the isolated strain clustered within Class II NDVs, and phylogenetically close to Genotype VI NDVs. The strain was highly
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Figure 1. Phylogenetic analysis of the complete genome of Pigeon Isolate Pigeon/China/SD069/2012 and Reference NDV. The phylogenetic tree was constructed by the neighbor-joining method with 1,000 bootstrap replicates (bootstrap values are shown on the tree).
CHARACTERIZATION OF NEWCASTLE DISEASE VIRUS ISOLATED FROM PIGEONS
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similar to pi/CH/LLN/110713 (99.78%), a PPMV-1 strain isolated in China. However, the isolated strain was genetically distinct and phylogenetically distant from the vaccine strains used in China (B1, LaSota, and Mukteswar). Genomic blast analysis revealed that the isolated strain had sequence homologies of 80.9, 80.8, and 84.2%, respectively, with Strain B1, LaSota, and Mukteswar.
Animal Experiments All the pigeons infected with SD069 exhibited had slight depression and head tremor at 2 dpi. At 4 dpi, all SD069 infected pigeons were severely depressed and had ruffled feathers. Additionally, some had severe nervous signs such as incoordination accompanied by leg paralysis (Figure 2A). By 5 dpi, one infected pigeon was dead, and altogether a total of 5 pigeons died within the 14-d observation period. At necropsy, brain hyperemia and hemorrhage were found (Figure 2B), and severe hemorrhage could be seen in the lung (Figure 2C). The gastrointestinal tract was empty in all dead pigeons, and multifocal hemorrhages were observed in the mucosa of the muscular stomach (Figure 2D) and small intestine (Figure 2E). Spleens were enlarged and mottled. Hemorrhaging of liver, trachea, and kidney could also be seen occasionally. No obvious clinical signs were seen in mock-infected pigeons within the 2-wk period. Histopathologically, hemorrhage, necrosis, and inflammation were observed in multiple organs. Necrosis of mucous epithelial cells were seen in the trachea
(Figure 3A). Severe bronchopneumonia was observed in the lungs. These changes were characterized by interstitial edema and extensive of lymphocytes, neutrophils, and infiltration of plasma cells around the bronchiolitis (Figure 3B). Pathological changes in the glandular stomach were hemorrhage and atrophy of the proventriculus papillae, with mucus on the surface of papillae. Necrosis of the mucosal epithelia of the proventriculus was also observed. Lesions in the small intestine of pigeons infected with SD069 virus showed signs of enteritis, characterized by broken villi, dropout of epithelium, and numerous inflammatory cells infiltrates (Figure 3C). The cardiac muscle fibers were elongated and thinned with unclear transverse striation, and some myocardial cells showed evidence of slight granular degeneration (Figure 3D). Capillaries in renal interstitium were congested, and typical granular degeneration occurred in the epithelial cells of convoluted tubule, some epithelial cells swelled and fell off to lumen (Figure 3E). A large number of lymphocytes were observed disrupting and disappearing in the spleen, and fibrin deposits were present in necrotic areas of the spleen (Figure 3F). The most remarkable histological finding was observed in the brain. Congestion in the blood vessels, endothelial cell hyperplasia, and central chromatolysis in the cerebral neurons were observed in all the virus-infected pigeons. Mononuclear inflammatory infiltrations in the neuropil, glial cell infiltration, and perivascular cuffing were also observed in some pigeons (Figure 3G, H). From immunohistochemistry, viral antigen was detected in bronchial epithelial cells (Figure 3I), neurons, and glial cells (Figure 3J). In comparison, all tissues samples
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Figure 2. Gross photographs in visceral organs from pigeons after intranasal inoculation with NDV SD069. (A) Pigeons infected exhibited severe nervous signs; (B) Brain hyperemia and hemorrhage; (C) Severe hemorrhage in the lung; (D) Multifocal hemorrhages in the mucosa of muscular stomach; (E) Multifocal hemorrhages in the mucosa of small intestine.
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Figure 3. Histopathology and immunohistochemical detection of NDV antigens on tissues from pigeons infected with NDV SD069. (A) Dropout and necrosis of mucous epithelial cells in the trachea (arrow); (B) Severe bronchopneumonia in the lung; (C) Dropout of broken villi, epithelium and numerous inflammatory cells infiltrates in the small intestine (arrow); (D) Cardiac muscle fibers showed granular degeneration (arrow); (E) Typical granular degeneration in the epithelial cells of convoluted tubule; (F) A large number of lymphocytes disrupting in the spleen, and fibrin deposits in necrotic areas; (G) and (H) Mononuclear cell infiltrates in the perivascular spaces (arrow) and neuronophagia (arrow); (I) Influenza virus antigen (arrow) in the epithelial cells of the bronchioles; (J) Influenza virus antigen (arrow) in neurons and glial cells.
CHARACTERIZATION OF NEWCASTLE DISEASE VIRUS ISOLATED FROM PIGEONS
from the control group had no apparent histological changes. Sera were collected at 14 dpi from surviving pigeons and HI tests were performed to detect specific NDV antibody. All sera were positive for NDV HI antibody after SD069 inoculation, and the average HI titers were 7.8 log 2. No obvious clinical signs were seen in chickens or ducks in the SD069 inoculation group within the 2-wk observation period. However, 14 d after inoculation of the virus, all chickens’ and ducks’ sera were positive for NDV HI antibody, and their average HI titers were 6 log 2 and 4.6 log 2, respectively.
DISCUSSION
allowed circulating among chickens, they could evolve to have a greater disease-causing capability. The potential for such an occurrence has already been realized for NDV. The 1998 to 2000 NDV outbreaks reported in Australia, with clinical disease and mortality observed among commercial chickens, were caused by a virulent NDV strain that evolved from a low-virulence strain that was apparently circulating among chickens for a long time (Gould et al., 2001). Vaccination has been widely used for prevention and control of the PPMV-1 disease in many countries including China, but the disease is still enzootic in pigeons in China (Liu et al., 2006; Yu et al., 2001). The most commonly used live vaccines LaSota and Clone-30 were close to Genotype II, while the prevailing PPMV1 strains belong to Genotype VI. There are significant differences between the prevailing NDV strains and the current vaccine strains in their biology, serology, and genetics, which might be considered as the reasons for the Newcastle disease outbreaks in pigeons in recent years. On the other hand, the nature of racing pigeon sport requires that birds from different breeders and different regions are brought together to compete in distance races. The gathering of birds and transportation stress often causes the vaccination failure. In summary, we isolated a PPMV-1 from a freeliving racing-pigeon flock and we demonstrated that the PPMV-1 caused severe lesions among infected pigeons, mostly affecting the brain. Although the virus did not cause disease in chickens and ducks, the virus could effectively infect and replicate in these poultry. Pigeons were considered seriously as a potential source of NDV infection and disease for commercial poultry flocks. Therefore, new vaccines to prevent the PPMV1 infection in the pigeon flocks should be developed as soon as possible, and strict biosecurity measures should be taken to reduce the risk of pigeon Newcastle disease outbreaks.
ACKNOWLEDGMENTS The study was supported by the National Natural Scientific Foundation (31172296).
REFERENCES Alexander, D. 1985. Avian paramyxovirus type 1 (NDV) infections in pigeons and poultry. Proc. Annu. West Poult. Conf., pp. 131– 134. Alexander, D., G. Parsons, and R. Marshall. 1984. Infection of fowls with Newcastle disease virus by food contaminated with pigeon faeces. Vet. Rec. 115:601–602. Chambers, P., N. S. Millar, R. W. Bingham, and P. T. Emmerson. 1986. Molecular cloning of complementary DNA to Newcastle disease virus, and nucleotide sequence analysis of the junction between the genes encoding the haemagglutinin-neuraminidase and the large protein. J. Gen. Virol. 67:475–486. Collins, M., J. Bashiruddin, and D. Alexander. 1993. Deduced amino acid sequences at the fusion protein cleavage site of Newcastle disease viruses showing variation in antigenicity and pathogenicity. Arch. Virol. 128:363–370.
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In this study, a PPMV-1 isolate recovered from a flock of racing pigeons in China that presented nervous signs was genotypically and pathotypically characterized. The strain had a genome size of 15,192 nt, has highly similar with PPMV-1 strains isolated in China, but was phylogenetically distant from the virus prevalence in chickens and vaccine strains used in China. This confirmed that the origin of this PPMV-1 was pigeon. Based on the current definition of the World Organization for Animal Health an NDV strain that either has an ICPI 0.7 or carries a typical velogenic amino acid motif at the F protein cleavage site can be classified as velogenic. However, the F proteins of the PPMV-1 strains examined in the study contain a polybasic cleavage site motif, but it caused no appearance disease and has middle ICPI in chickens. This indicates that the virulence of NDV strains can be qualified rather than quantified by the analysis of cleavage site motifs, and that pathogenicity tests such as MDT and ICPI cannot be replaced by analysis of the cleavage site to precisely characterize the virulence and pathogenicity of NDV strains. Furthermore, other protein precursors also play a role in the virulence of NDVs (Dortmans et al., 2011). In pigeon pathogenicity experiments, the most remarkable and consistent histopathologic changes were observed in the brain, and abundant viral antigens were observed in the brain of PPMV-1-infected birds. Therefore the virus tropism for brain and the brain lesions account for the severe nervous signs and highly mortality observed in the racing pigeons flock. Although this PPMV-1 strain caused no appearance disease in chickens and ducks, it could effective infected of the poultry as evidence of seroconversion, and previous investigators demonstrated that PPMV-1 could enhance virulence for chickens when serially passage through chickens (Dortmans et al., 2011; Kommers et al., 2003). Another concern regarding PPMV-1 is its ability to spread from infected pigeons to nearby chickens. As racing pigeons have free-flying characteristics, they come into frequent contact with wild birds and domestic poultry. In one study, the contact infection rate of 2 different PPMV-1 strains in 4-week-old SPF chickens reached 100% (Kissi, 1988). If these PPMV-1 strains
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LIU ET AL. Liu, H., Z. Wang, C. Song, Y. Wang, B. Yu, D. Zheng, C. Sun, and Y. Wu. 2006. Characterization of pigeon-origin Newcastle disease virus isolated in China. Avian Dis. 50:636–640. Mayo, M. 2002. A summary of taxonomic changes recently approved by ICTV. Arch. Virol. 147:1655–1656. Nagai, Y., H. D. Klenk, and R. Rott. 1976. Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. Virology 72:494–508. Ogasawara, T., B. Gotoh, H. Suzuki, J. Asaka, K. Shimokata, R. Rott, and Y. Nagai. 1992. Expression of Factor X and its significance for the determination of paramyxovirus tropism in the chick embryo. EMBO J. 11:467–472. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty per cent endpoints. Am. J. Epidemiol. 27:493–497. Rui, Z., P. Juan, S. Jingliang, Z. Jixun, W. Xiaoting, Z. Shouping, L. Xiaojiao, and Z. Guozhong. 2010. Phylogenetic characterization of Newcastle disease virus isolated in the mainland of China during 2001–2009. Vet. Microbiol. 141:246–257. Vindevogel, H., and J. Duchatel. 1988. Panzootic Newcastle disease virus in pigeons. Newcastle Dis. 8:184–196. Yu, L., Z. Wang, Y. Jiang, L. Chang, and J. Kwang. 2001. Characterization of newly emerging Newcastle disease virus isolates from the People’s Republic of China and Taiwan. J. Clin. Microbiol. 39:3512–3519. Zhang, S., X. Wang, C. Zhao, D. Liu, Y. Hu, J. Zhao, and G. Zhang. 2011. Phylogenetic and pathotypical analysis of two virulent Newcastle disease viruses isolated from domestic ducks in China. PLoS ONE. 6:e25000.
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Dortmans, J. C., P. Rottier, G. Koch, and B. P. Peeters. 2011. Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence. J. Gen. Virol. 92:336–345. Gould, A. R., J. A. Kattenbelt, P. Selleck, E. Hansson, A. DellaPorta, and H. A. Westbury. 2001. Virulent Newcastle disease in Australia: Molecular epidemiological analysis of viruses isolated prior to and during the outbreaks of 1998–2000. Virus Res. 77:51– 60. King, D. 1996. Avian paramyxovirus Type 1 from pigeons: Isolate characterization and pathogenicity after chicken or embryo passage of selected isolates. Avian Dis. 707–714. Kissi, B. 1988. Studies on the virulence of pigeon paramyxovirus1 (PMV-1). I. Changes in the virulence of pigeon PMV-1 strains isolated in Hungary upon passage in chickens, embryonated hen’s eggs and pigeons. Acta Vet. Hung. 36:283–92. Kommers, G. D., D. J. King, B. S. Seal, and C. C. Brown. 2003. Virulence of six heterogeneous-origin Newcastle disease virus isolates before and after sequential passages in domestic chickens. Avian Pathol. 32:81–93. Lana, D., D. Snyder, D. King, and W. Marquardt. 1988. Characterization of a battery of monoclonal antibodies for differentiation of Newcastle disease virus and pigeon paramyxovirus-1 strains. Avian Dis. 32:273–281. Lister, S., D. Alexander, and R. Hogg. 1986. Evidence for the presence of avian paramyxovirus Type 1 in feral pigeons in England and Wales. Vet. Rec. 118:476–479.
Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China; and † College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian 271018, China fusion (F) protein confirmed that the isolate contained the virulent motif 112 RRQKRF117 at the cleavage site, but it caused no appearance disease in chickens and ducks. However, the isolate had virulence in pigeons, resulting in severe nervous signs and highly mortality. Pigeons were considered as a potential source of NDV infection and disease for commercial poultry flocks. Therefore, new vaccines to prevent the NDV infection in the pigeon flocks should be developed as soon as possible, and strict biosecurity measures should be taken to reduce the risk of pigeon Newcastle disease outbreaks.
Key words: Newcastle disease virus, racing pigeon, phylogenetic analysis, pathology 2015 Poultry Science 94:1476–1482 http://dx.doi.org/10.3382/ps/pev106
INTRODUCTION Newcastle disease is a highly contagious viral disease of poultry and other bird species caused by the Newcastle disease virus (NDV), a member of the genus Avulavirus in the family Paramyxoviridae (Mayo, 2002; Nagai et al., 1976). The genome of NDV is a nonsegmented, single-stranded, negative-sense RNA molecule of approximately 15,186 nucleotides (nt) that contains 6 genes encoding the 6 structural proteins (from the 3 to 5 terminus): nucleoprotein, phosphoprotein, matrix, fusion (F), hemagglutinin-neuraminidase (HN), and the large protein (Chambers et al., 1986). NDV strains can be classified as high virulence (velogenic), intermediate (mesogenic), or low virulence (lentogenic) based on various biological parameters, such as the mean death time (MDT) of chicken embryos infected with the minimum lethal dose of virus, the intracerebral pathogenicity index (ICPI) in 1-day-old chicks, and the intravenous pathogenicity index (IVPI) in 6week-old chickens. The primary molecular determinant C 2015 Poultry Science Association Inc. Received December 4, 2014. Accepted February 27, 2015. 1 Corresponding author: [email protected]
for NDV pathogenicity is the amino acids of the F protein cleavage site (Nagai et al., 1976; Ogasawara et al., 1992). NDV has a wide host range with most orders of birds that reported to have been infected by the virus. More commonly affected species include chickens, turkeys, pigeons, and ducks (King, 1996; Lana et al., 1988; Zhang et al., 2011). Variant strains of NDV are associated with infections of pigeons, known as pigeon paramyxovirus Type 1 (PPMV-1), which can be characterized by unique monoclonal-antibody-binding profiles (King, 1996; Lana et al., 1988). After its first emergence in the Middle East (late 1970s) (Lister et al., 1986), PPMV-1 spread worldwide (Alexander, 1985; Lister et al., 1986; Vindevogel and Duchatel, 1988), and several Newcastle disease outbreaks in chickens have been attributed to PPMV-1 (Alexander et al., 1984). Therefore PPMV-1 presents which makes it a real threat to the poultry industry. The frequent isolation of PPMV-1 from pigeons across China necessitates an assessment of the risk potential of these viruses causing disease in poultry (Liu et al., 2006; Yu et al., 2001). In the present study, a PPMV-1 isolate from outbreaks in racing pigeons in China was pathotypically and genotypically characterized.
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ABSTRACT A Newcastle disease virus (NDV) isolated from an outbreak in racing pigeons in China was characterized in this study. Complete gene of the NDV isolate was sequenced and phylogenetic analysis. Pathogenicity experiment was carried out in pigeons, chickens, and ducks. Phylogenetic analysis revealed that the strain clustered with the Class II viruses, has highly phylogenetically similar to NDV strains isolated from pigeons in China, but was distant from the viruses prevalence in chickens and vaccine strains used in China. The deduced amino acid sequence of the cleavage site of the
CHARACTERIZATION OF NEWCASTLE DISEASE VIRUS ISOLATED FROM PIGEONS
MATERIALS AND METHODS Virus Isolation and Identification
isolate, virus-infective allantoic fluid was diluted 10fold in saline from 10−1 to 10−8 . For each dilution, 3 eggs were used to inoculate 0.1 mL diluted virus via the allantoic cavity. After 72 h, virus infection was assessed by HA assay of egg allantoic fluid. EID50 was calculated using the method of Reed and Muench (1938).
Phylogenetic Analysis and Molecular Characterization Viral RNA was extracted using RNeasy Mini Kit (Qiagen, Chatsworth, CA). reverse transcription and PCR was performed under standard conditions as previously described (Liu et al., 2006). Primer sequences used to generate for the overlapping subgenomic cDNA fragments and for genome sequencing are available upon request. PCR products were purified using the QIA quick PCR Purification Kit (Qiagen, Valencia, CA), and then sequenced by the Beijing BGI-GBI Biotech Co., Ltd. Reference NDV genomic sequences were obtained from GenBank, and these included current vaccine strains, typical prevailing isolates in China, and the reference strains for each known NDV genotype. These NDV sequences and the complete coding sequences of the NDV isolate were aligned and analyzed using the ClustalW multiple alignment algorithm in the MegAlign program of the DNASTAR software suite (version 3.1; DNAstar, Madison, WI). Phylogenetic analysis was performed using software MEGA 4.1. (DNAStar Inc.) by the distance-based neighbourjoining method (1,000 replicates for bootstrap).
Animals To investigate the biological characteristics and pathogenicity of the NDV isolate, SPF chickens, NDV antibody-negative pigeons, and NDV antibody-negative Beijing ducks were used. All animal research was approved by Beijing Administration Committee of Laboratory Animals under the leadership of the Beijing Association for Science and Technology; the approve identifier is SYXK (Beijing) 2007–0023.
Assessment of the Biological Characteristics of the Isolate The pathogenic potential for the isolated viruses was evaluated using standard assay methods to determine the MDT in 10-day-old chick embryos, the ICPI in 1-day-old chickens, and IVPI in 6-weeks-old chickens. For MDT test, virus-infective allantoic fluid was diluted 10-fold in saline from 10−6 to 10−10 . For each dilution, 5 eggs were used to inoculate 0.2 mL diluted virus via the allantoic cavity. After 24 h, the eggs were examined for mortality at 2-h intervals for 120 h. The MDT was the mean time in hours for the lowest dilution to kill all eggs of that dilution. To test the 50% embryo infectious doses (EID50 ) of the
Animal Experiments Twenty 1-month-old NDV antibody-negative pigeons, twenty 6-weeks-old SPF chickens, and twenty 2weeks-old NDV antibody-negative Peking ducks were randomly divided into 2 groups, respectively. The challenge procedure was birds were inoculated via the intranasal route with 106 EID50 of the virus in a 0.2 mL volume or 0.2 mL PBS as a mock-infected control. All birds were clinically monitored every day for signs of disease (e.g., disheveled feathers, lethargy, fever or paralysis) and mortality. Dead birds were immediately necropsied for the determination of gross lesions, and their tissue samples (trachea, lung, brain, spleen, small intestine, proventriculus, and kidney) were collected and fixed in 10% neutral buffered formalin for histological observation. In addition, antigens of NDV present in the tissue of virus inoculation birds were further examined using immunohistochemistry, which employed an HN-protein-specific mouse monoclonal antibody. Serum samples were collected from all birds before inoculation and at 14 d postinfection (dpi) for NDV-specific antibody detection using an HI test with 1% chicken red blood cells.
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In February 2012, a flock of racing pigeons displayed severe nervous signs in Shandong Province of China. Tracheal and cloacal swabs were collected from the diseased pigeons. Swabs samples were maintained in transport medium containing antibiotics and kept at 4◦ C during transportation to the laboratory. Samples were centrifuged for 10 min at 3,000 × g at 4◦ C and 0.2 mL supernatant was used to inoculate the allantoic cavities of 9- to 11-day-old specific pathogen-free (SPF) embryonated chicken eggs, and incubated at 35◦ C for 72 h. Allantoic fluids were then harvested and tested for haemagglutinin (HA) activity. Positive isolates were characterized by HA inhibition (HI) tests using NDV, H5, H9 subtype avian influenza virus, and egg drop syndrome virus antibodies. The pigeon virus showed HA activity, which was inhibited by NDV antibody, but revealed no cross-reaction to avian influenza virus and egg drop syndrome virus antibodies in HI test. The strain was designated Pigeon/China/SD069/2012. To purify the virus, 10-fold dilutions of a virus stock are prepared, and 0.1-mL aliquots are inoculated onto baby hamster kidney cell monolayers. After an incubation period, the monolayers are covered with a nutrient agar. After 48 h, each infectious particle produces a plaque. The virus was purified 3 times using a plaque technique before being propagated in the allantoic cavities of 10-dayold SPF embryonated chicken eggs. Virus stocks were stored at −80◦ C until use.
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RESULTS Biological Characteristic Assessment of the SD069 Isolate Initial biological characterization of the isolate strain SD069 included MDT, ICPI, and IVPI were determinations. The virus exhibited MDT of 96 h and the ICPI values were 0.6 for the moderately virulent isolate. The virus did not produce obvious signs or lesions in all the 10 chickens within 10-d obvious period, indicating the IVPI was 0.
Sequence and Phylogenetic Analysis The nucleotide sequence data were deposited into the Genbank database and the accession numbers was
KP861633. The total length of the strain was 15,192 nt. Compared with the NDV Lasota strain, the isolated strain bears a 6-nt insertion (TCCCCA) in positions 1,647 to 1,648 nt of the nucleoprotein gene. Proteolytic cleavage site motifs (Residues 112 to 117) for the F0 protein in the isolate were analyzed. The strain has a virulent motif (112 RRQKRF117 ) composed of multibasic amino acids at the precursor F (F0) cleavage site. This motif is commonly found in strains that are highly virulent in chickens (Collins et al., 1993; Rui et al., 2010). Phylogenetic analysis was conducted based on complete nucleotide sequences. Nucleotide sequence data of 32 NDV reference strains obtained from the GenBank database were used for comparison. As shown in Figure 1, the isolated strain clustered within Class II NDVs, and phylogenetically close to Genotype VI NDVs. The strain was highly
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Figure 1. Phylogenetic analysis of the complete genome of Pigeon Isolate Pigeon/China/SD069/2012 and Reference NDV. The phylogenetic tree was constructed by the neighbor-joining method with 1,000 bootstrap replicates (bootstrap values are shown on the tree).
CHARACTERIZATION OF NEWCASTLE DISEASE VIRUS ISOLATED FROM PIGEONS
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similar to pi/CH/LLN/110713 (99.78%), a PPMV-1 strain isolated in China. However, the isolated strain was genetically distinct and phylogenetically distant from the vaccine strains used in China (B1, LaSota, and Mukteswar). Genomic blast analysis revealed that the isolated strain had sequence homologies of 80.9, 80.8, and 84.2%, respectively, with Strain B1, LaSota, and Mukteswar.
Animal Experiments All the pigeons infected with SD069 exhibited had slight depression and head tremor at 2 dpi. At 4 dpi, all SD069 infected pigeons were severely depressed and had ruffled feathers. Additionally, some had severe nervous signs such as incoordination accompanied by leg paralysis (Figure 2A). By 5 dpi, one infected pigeon was dead, and altogether a total of 5 pigeons died within the 14-d observation period. At necropsy, brain hyperemia and hemorrhage were found (Figure 2B), and severe hemorrhage could be seen in the lung (Figure 2C). The gastrointestinal tract was empty in all dead pigeons, and multifocal hemorrhages were observed in the mucosa of the muscular stomach (Figure 2D) and small intestine (Figure 2E). Spleens were enlarged and mottled. Hemorrhaging of liver, trachea, and kidney could also be seen occasionally. No obvious clinical signs were seen in mock-infected pigeons within the 2-wk period. Histopathologically, hemorrhage, necrosis, and inflammation were observed in multiple organs. Necrosis of mucous epithelial cells were seen in the trachea
(Figure 3A). Severe bronchopneumonia was observed in the lungs. These changes were characterized by interstitial edema and extensive of lymphocytes, neutrophils, and infiltration of plasma cells around the bronchiolitis (Figure 3B). Pathological changes in the glandular stomach were hemorrhage and atrophy of the proventriculus papillae, with mucus on the surface of papillae. Necrosis of the mucosal epithelia of the proventriculus was also observed. Lesions in the small intestine of pigeons infected with SD069 virus showed signs of enteritis, characterized by broken villi, dropout of epithelium, and numerous inflammatory cells infiltrates (Figure 3C). The cardiac muscle fibers were elongated and thinned with unclear transverse striation, and some myocardial cells showed evidence of slight granular degeneration (Figure 3D). Capillaries in renal interstitium were congested, and typical granular degeneration occurred in the epithelial cells of convoluted tubule, some epithelial cells swelled and fell off to lumen (Figure 3E). A large number of lymphocytes were observed disrupting and disappearing in the spleen, and fibrin deposits were present in necrotic areas of the spleen (Figure 3F). The most remarkable histological finding was observed in the brain. Congestion in the blood vessels, endothelial cell hyperplasia, and central chromatolysis in the cerebral neurons were observed in all the virus-infected pigeons. Mononuclear inflammatory infiltrations in the neuropil, glial cell infiltration, and perivascular cuffing were also observed in some pigeons (Figure 3G, H). From immunohistochemistry, viral antigen was detected in bronchial epithelial cells (Figure 3I), neurons, and glial cells (Figure 3J). In comparison, all tissues samples
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Figure 2. Gross photographs in visceral organs from pigeons after intranasal inoculation with NDV SD069. (A) Pigeons infected exhibited severe nervous signs; (B) Brain hyperemia and hemorrhage; (C) Severe hemorrhage in the lung; (D) Multifocal hemorrhages in the mucosa of muscular stomach; (E) Multifocal hemorrhages in the mucosa of small intestine.
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Figure 3. Histopathology and immunohistochemical detection of NDV antigens on tissues from pigeons infected with NDV SD069. (A) Dropout and necrosis of mucous epithelial cells in the trachea (arrow); (B) Severe bronchopneumonia in the lung; (C) Dropout of broken villi, epithelium and numerous inflammatory cells infiltrates in the small intestine (arrow); (D) Cardiac muscle fibers showed granular degeneration (arrow); (E) Typical granular degeneration in the epithelial cells of convoluted tubule; (F) A large number of lymphocytes disrupting in the spleen, and fibrin deposits in necrotic areas; (G) and (H) Mononuclear cell infiltrates in the perivascular spaces (arrow) and neuronophagia (arrow); (I) Influenza virus antigen (arrow) in the epithelial cells of the bronchioles; (J) Influenza virus antigen (arrow) in neurons and glial cells.
CHARACTERIZATION OF NEWCASTLE DISEASE VIRUS ISOLATED FROM PIGEONS
from the control group had no apparent histological changes. Sera were collected at 14 dpi from surviving pigeons and HI tests were performed to detect specific NDV antibody. All sera were positive for NDV HI antibody after SD069 inoculation, and the average HI titers were 7.8 log 2. No obvious clinical signs were seen in chickens or ducks in the SD069 inoculation group within the 2-wk observation period. However, 14 d after inoculation of the virus, all chickens’ and ducks’ sera were positive for NDV HI antibody, and their average HI titers were 6 log 2 and 4.6 log 2, respectively.
DISCUSSION
allowed circulating among chickens, they could evolve to have a greater disease-causing capability. The potential for such an occurrence has already been realized for NDV. The 1998 to 2000 NDV outbreaks reported in Australia, with clinical disease and mortality observed among commercial chickens, were caused by a virulent NDV strain that evolved from a low-virulence strain that was apparently circulating among chickens for a long time (Gould et al., 2001). Vaccination has been widely used for prevention and control of the PPMV-1 disease in many countries including China, but the disease is still enzootic in pigeons in China (Liu et al., 2006; Yu et al., 2001). The most commonly used live vaccines LaSota and Clone-30 were close to Genotype II, while the prevailing PPMV1 strains belong to Genotype VI. There are significant differences between the prevailing NDV strains and the current vaccine strains in their biology, serology, and genetics, which might be considered as the reasons for the Newcastle disease outbreaks in pigeons in recent years. On the other hand, the nature of racing pigeon sport requires that birds from different breeders and different regions are brought together to compete in distance races. The gathering of birds and transportation stress often causes the vaccination failure. In summary, we isolated a PPMV-1 from a freeliving racing-pigeon flock and we demonstrated that the PPMV-1 caused severe lesions among infected pigeons, mostly affecting the brain. Although the virus did not cause disease in chickens and ducks, the virus could effectively infect and replicate in these poultry. Pigeons were considered seriously as a potential source of NDV infection and disease for commercial poultry flocks. Therefore, new vaccines to prevent the PPMV1 infection in the pigeon flocks should be developed as soon as possible, and strict biosecurity measures should be taken to reduce the risk of pigeon Newcastle disease outbreaks.
ACKNOWLEDGMENTS The study was supported by the National Natural Scientific Foundation (31172296).
REFERENCES Alexander, D. 1985. Avian paramyxovirus type 1 (NDV) infections in pigeons and poultry. Proc. Annu. West Poult. Conf., pp. 131– 134. Alexander, D., G. Parsons, and R. Marshall. 1984. Infection of fowls with Newcastle disease virus by food contaminated with pigeon faeces. Vet. Rec. 115:601–602. Chambers, P., N. S. Millar, R. W. Bingham, and P. T. Emmerson. 1986. Molecular cloning of complementary DNA to Newcastle disease virus, and nucleotide sequence analysis of the junction between the genes encoding the haemagglutinin-neuraminidase and the large protein. J. Gen. Virol. 67:475–486. Collins, M., J. Bashiruddin, and D. Alexander. 1993. Deduced amino acid sequences at the fusion protein cleavage site of Newcastle disease viruses showing variation in antigenicity and pathogenicity. Arch. Virol. 128:363–370.
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In this study, a PPMV-1 isolate recovered from a flock of racing pigeons in China that presented nervous signs was genotypically and pathotypically characterized. The strain had a genome size of 15,192 nt, has highly similar with PPMV-1 strains isolated in China, but was phylogenetically distant from the virus prevalence in chickens and vaccine strains used in China. This confirmed that the origin of this PPMV-1 was pigeon. Based on the current definition of the World Organization for Animal Health an NDV strain that either has an ICPI 0.7 or carries a typical velogenic amino acid motif at the F protein cleavage site can be classified as velogenic. However, the F proteins of the PPMV-1 strains examined in the study contain a polybasic cleavage site motif, but it caused no appearance disease and has middle ICPI in chickens. This indicates that the virulence of NDV strains can be qualified rather than quantified by the analysis of cleavage site motifs, and that pathogenicity tests such as MDT and ICPI cannot be replaced by analysis of the cleavage site to precisely characterize the virulence and pathogenicity of NDV strains. Furthermore, other protein precursors also play a role in the virulence of NDVs (Dortmans et al., 2011). In pigeon pathogenicity experiments, the most remarkable and consistent histopathologic changes were observed in the brain, and abundant viral antigens were observed in the brain of PPMV-1-infected birds. Therefore the virus tropism for brain and the brain lesions account for the severe nervous signs and highly mortality observed in the racing pigeons flock. Although this PPMV-1 strain caused no appearance disease in chickens and ducks, it could effective infected of the poultry as evidence of seroconversion, and previous investigators demonstrated that PPMV-1 could enhance virulence for chickens when serially passage through chickens (Dortmans et al., 2011; Kommers et al., 2003). Another concern regarding PPMV-1 is its ability to spread from infected pigeons to nearby chickens. As racing pigeons have free-flying characteristics, they come into frequent contact with wild birds and domestic poultry. In one study, the contact infection rate of 2 different PPMV-1 strains in 4-week-old SPF chickens reached 100% (Kissi, 1988). If these PPMV-1 strains
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Dortmans, J. C., P. Rottier, G. Koch, and B. P. Peeters. 2011. Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence. J. Gen. Virol. 92:336–345. Gould, A. R., J. A. Kattenbelt, P. Selleck, E. Hansson, A. DellaPorta, and H. A. Westbury. 2001. Virulent Newcastle disease in Australia: Molecular epidemiological analysis of viruses isolated prior to and during the outbreaks of 1998–2000. Virus Res. 77:51– 60. King, D. 1996. Avian paramyxovirus Type 1 from pigeons: Isolate characterization and pathogenicity after chicken or embryo passage of selected isolates. Avian Dis. 707–714. Kissi, B. 1988. Studies on the virulence of pigeon paramyxovirus1 (PMV-1). I. Changes in the virulence of pigeon PMV-1 strains isolated in Hungary upon passage in chickens, embryonated hen’s eggs and pigeons. Acta Vet. Hung. 36:283–92. Kommers, G. D., D. J. King, B. S. Seal, and C. C. Brown. 2003. Virulence of six heterogeneous-origin Newcastle disease virus isolates before and after sequential passages in domestic chickens. Avian Pathol. 32:81–93. Lana, D., D. Snyder, D. King, and W. Marquardt. 1988. Characterization of a battery of monoclonal antibodies for differentiation of Newcastle disease virus and pigeon paramyxovirus-1 strains. Avian Dis. 32:273–281. Lister, S., D. Alexander, and R. Hogg. 1986. Evidence for the presence of avian paramyxovirus Type 1 in feral pigeons in England and Wales. Vet. Rec. 118:476–479.