Background
Newcastle disease (ND) in pigeons is responsible for one of the most severe recent infectious pigeon diseases in China [
1‐
3]. The causative agent of ND in pigeons is Newcastle disease virus (NDV), also called pigeon paramyxovirus type 1 (PPMV-1). NDV is taxonomically classified into the genus
Avulavirus, subfamily
Paramyxovirinae, family
Paramyxoviridae, order
Mononegaviriales [
4,
5]. The NDV genome consists of a single-stranded, negative-sense RNA, containing six genes in the sequence of 3′-NP-P-M-F-HN-L-5′. Six structural proteins are encoded as nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN) and large protein (L) [
6]. In addition, two nonstructural proteins, V and W, are generated from the P gene by RNA editing [
7,
8].
NDV isolates are phylogenetically divided into two major classes, class I and class II [
9,
10]. Class I NDV isolates are widely separated from healthy birds, and nearly all are nonvirulent [
10‐
14]. NDVs belonging to class I are characterized by a 15,198-nt genomic RNA. By contrast, class II NDVs have two genome types. According to the classification system proposed by Claudio L. Afonso et al. in 2012, class I viruses contain a single genotype whereas class II NDV isolates can be effectively divided in 18 genotypes (I - XVIII) [
15]. Genotype I-IV viruses are early lineage (before 1960) with a genome size of 15,186 nt. Genotype V-VIII are recent lineage (after 1960) with a genome of 15,192 nt [
4,
10,
16‐
18]. Most pigeon-derived virulent NDV isolates are class II genotype VI, which was further divided into VIa - VIg. Subtype VIa strains, with the representative strain Iraq AG68, were isolated from Middle Eastern epizootics in the 1960s and subsequently spread to Africa and Asian countries [
19]. Subtype VIb NDV of pigeon origin is widely accepted to be responsible for the third ND pandemic during the 1980s. In a Chinese outbreak around 2000, subgenotype VIb [
20], VIf and VIg [
19] were all identified from pigeons, chickens and other fowls, whereas in the last decade, little VIf and VIg outbreaks have been reported [
21].
Based on the pathogenicity of ND strains in chickens, NDV isolates are generally classified as lentogenic (nonvirulent), mesogenic (intermediate virulent) and velogenic (highly virulent), which are further classified as viscerotropic velogenic and neurotropic velogenic [
22]. The virulence of NDV strains is reported to be mainly determined by cleavage site sequence of the F protein [
23,
24]. NDV F protein is generated as precursor F0; when cleaved into disulfide-linked F1 and F2 polypeptides, F protein changes to be fusogenic and is crucial in mediating fusion of the viral envelope with cellular membranes. The consensus sequence of F protein cleavage sites of velogenic and mesogenic strains is
112(R/K)RQ(R/K)RF
117. Common lenogenic strains have a monobasic amino acid motif at the same site,
112(G/E)(K/R)Q(G/E)R↓L
117. Different F protein cleavage sites are substrates for different types of cellular proteases [
25,
26]. The F protein of lentogenic strains is cleaved only by trypsin-like enzymes, which are confined to the respiratory and intestinal tracts. The F protein of virulent viruses such as genotype VI and VII strains are cleaved by ubiquitous and diverse host protease(s). Based on these findings, virulent NDV is defined by the World Organization for Animal Health as a virus of avian paramyxovirus-1 (APMV-1) with at least three multiple basic amino acids (arginine or lysine) residues at the C-terminus of the F2 protein (between residues 113 and 116) and phenylalanine at residue 117, which is the N-terminus of the F1 protein [
22,
27].
For many years, ND has been one of the most important infectious pigeon diseases in China, but low mortality of this disease is widely reported in most countries. After infection with pigeon-origin genotype VI ND, pigeons showed clinical signs similar to neurotropic ND-infected chickens, with a morbidity from 30% to 70% and a mortality no more than 10% [
28,
29]. In recent years, a high mortality has been observed in ND-infected pigeons in China. Mortality is from 40% to 80% or 100% in some cases [
1‐
3]. Whether pigeon-origin NDV isolates in China are more pathogenic for pigeons remains unclear. In this study, we analyzed genomic sequences of four PPMV-1 strains recently isolated in China, furthermore, the virulence, antigenicity and potential threat of the viruses were evaluated.
Methods
Viruses and pathogenicity tests
Pigeon-origin NDV strains pi/SD/CH/0132/2012 (designated ND132 in this study), pi/SH/CH/0163/2012 (ND163), pi/SH/CH/0167/2013 (ND167), and pi/SH/CH/0168/2013 (ND168) were isolated from clinical specimens of diseased pigeons in Shanghai and Shandong Provinces in China in 2012 and 2013. F48E8 (eighth egg-passaged stock of F48), the standard ND challenge strain in China [
19] and chicken vaccine strain LaSota were from the China Institute of Veterinary Drug Control (Beijing, China). All viruses were propagated in 9- to 11-day-old specific-pathogen-free (SPF) chicken embryonated eggs as described [
30]. Fresh allantoic fluid was harvested from embryonated eggs dead between 24 and 120 h after inoculation, and kept at −80 °C. Mean death time (MDT) and intracerebral pathogenicity index (ICPI) tests were determined as previously described to determine the virulence of the four isolates [
27].
The genomic RNA of ND132, ND163, ND167, and ND168 was extracted from the allantoic fluid of infected eggs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. RNA pellets were resuspended in 50 μL RNase-free water and reverse transcribed with 6-nt random primer with Mo-MLV reverse transcriptase (Promega, Madison, WI, USA). The cDNA was used as template to generate nine successive and overlapping DNA fragments by PCR using primer pairs specific for genotype VI NDV strains (Additional file
1: Table S1). Reverse transcription and PCR was performed without modification as described [
31].
To determine the 3′- and 5′-ends of the viral genomes, rapid amplification of cDNA end (RACE) was performed as reported previously [
32]. For 3′-RACE, genomic RNA was ligated with 5′-end-phosphorylated adaptor CL+ (Additional file
1: Table S1) using T4 RNA ligase (Fermentas, Shenzhen, China) in the presence of RNasin (Takara Biotechnology, Dalian, China) at 1 U/μl. After denaturation at 75 °C for 15 min, ligated RNA was transcribed with anti-adaptor CL- using Mo-MLV Reverse Transcriptase, which was complementary to adaptor primer CL+. The 3′-end of the genome was PCR-amplified with CL- and 3TSR, which was specific for the NP gene.
For 5′-RACE, first-strand cDNA was reverse-transcribed from viral RNA using specific primer 5TLF. After treatment with an equal volume of 0.6 N NaOH for 20 min at 60 °C to hydrolyze mRNA, cDNA was ligated with adaptor CL+ using T4 RNA ligase. Adaptor-ligated cDNA was PCR-amplified using primer 5TLF and anti-adaptor primer CL- and 1 μl PCR products was used for a second-round hemi-nested PCR reaction with primer CL- and specific primer 5TSF to generate the 5′ end of the viral genome.
PCR products were extracted from agarose gels and ligated into the TA cloning system (Promega). Ligation products were transferred into Escherichia coli DH5α and identified by restriction enzyme analysis. At least five clones for each segment were sent to Sangon Biotechnology (Shanghai, China) for sequencing.
Sequence analysis
Prediction of amino acid sequences, alignment of sequences and phylogenetic analysis was performed using the MegAlign program in the Lasergene package (DNASTAR Inc. Madison, WI, USA). Phylogenetic trees were constructed using MEGA version 7 [
33]. The genome sequences of 37 reference NDV strains, and the F gene sequences from 360 genotype VI NDV isolates and 24 other genotype representative strains were from EMBL/GenBank [
31,
34]. All data and accession numbers of NDV strains used in this study are listed in Additional file
2: Table S2.
Cross hemagglutination inhibition and cross virus-neutralization tests
Cross hemagglutination inhibition test was performed as described [
3] using four hemagglutinating units of LaSota, ND167 or ND132 virus and antisera against the NDV strains. For cross virus-neutralization (VN) tests, the 50% embryo infectious dose (EID
50) value of LaSota, ND167 and ND132 viruses was determined in advance by inoculation at a 10-fold dilution into 10-day-old SPF embryonated chicken eggs. Antisera against LaSota, ND167 and ND132 were serially double-diluted and mixed respectively with each NDV strain at 100 EID
50/100 μl. After incubation at 4 °C for 1 h, mixtures were injected into five, 10-day-old SPF embryonated chicken eggs to determine neutralization levels. Results of cross-VN and cross-HI tests were expressed as average and standard deviation (SD) and
R-value was calculated using the formula of Archetti and Horsfall [
35].
Antisera used in this study were obtained from chickens and pigeons according to a previous report [
1], with minor modification. First, 30-day-old SPF chickens and 50-day-old clinically healthy pigeons were intramuscularly injected with inactive oil-emulsified LaSota, ND167 or ND132 strains (10
6 EID
50 per chick and 10
5 EID
50 per pigeon). After 2 weeks, booster doses were administered. Birds were exsanguinated for serum collection 1 week after last inoculation. All sera were inactivated at 56 °C for 30 min and stored at −80 °C.
Animal experiment design
A total of 56 clinically healthy 40-day-old pigeons without any antibodies against ND were used and housed in negative pressure isolators (Fengshi Group, Suzhou, China). Experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Samples were collected from three pigeons randomly chosen before experiments and all pigeons after experiments were detected for other pigeon viruses by multiplex PCR [
36]. All pigeons were free of pigeon herpesvirus, adenovirus and circovirus (data not shown).
Pigeons were randomly divided into eight groups of seven birds per group: groups A1, A2, A3, B1, B2, B3, PC and NC. All pigeons were inoculated with 0.2 ml 106 EID50 virus via intravenous injection (groups A1 and B1), intramuscular injection (groups A2 and B2) or oral administration (groups A3 and B3). The titers of undiluted virus stock of ND132, ND167 and F48E8 were determined as 107.3 ELD50/0.2 ml, 107.9 ELD50/0.2 ml and 108.3 ELD50/0.2 ml respectively. Pigeons in a positive control (PC) group were intramuscularly injected with 106 EID50 F48E8; pigeons in negative control (NC) group were inoculated with sterile PBS (pH 7.2). Immediately after virus inoculation, all pigeons were kept in negative pressure isolators (Fengshi Group), under controlled laboratory and biosafety conditions. Pigeons were observed twice a day for 42 days, and any abnormal posture or behavior was recorded. Oral and cloacal swabs were collected at 5, 7, 10, 14, 21, 28, 35 and 42 days post infection (dpi) from experimental pigeons. Collected swabs were placed in 1.0 ml sterile PBS pH 7.2 and kept in −80 °C. Virus shedding was determined by virus isolation from swabs, using three SPF chicken eggs per sample.
Discussion
Four virulent PPMV-1 strains, ND132, ND163, ND167 and ND168, were isolated in Chinese pigeon farms; 70–80% of raised pigeons died from the outbreaks [
41]. The genomes of the viruses were sequenced and subjected to phylogenetic analysis with sequences from 380 NDV strains isolated from China, Japan, the Middle East, Europe and North America. Based on analysis of six viral proteins, all four isolates were classified into subgenotype VIb and had high homology to most PPMV-1 strains isolated after 2000 from distinct provinces in China (Fig.
2, Additional file
3: Table S3 and Additional file
4: Table S4). In a Chinese outbreak around 2000, subgenotype VIb, VIf and VIg [
19] were all identified from pigeons, chickens and other fowls, whereas in the last decade, little VIf and VIg outbreaks have been reported [
21]. Subgenotype VIb NDV is the most common in pigeons worldwide in the past two decades, and isolation of other subgenotypes, such as VIa, VId, VIf and VIg is rarely reported. Our four PPMV-1 isolates, ND132, ND163, ND167 and ND168, had high homology to each other but not to Chinese VIb isolates (representative strain YZ9712) from 10 years ago.
In this study, we used the Aldous method to further classify genotype VI genotype strains, which was based on 374 nucleotides in F protein of PPMV-1 isolated from Asia, Europe and America from 1978 to 2002 [
34]. Based on this typing method, subgenotype VIb was divided into six clades: a, b, c, d, e and f. The first three were termed 4bi and the others 4bii. Two groups of VIb PPMV-1 were frequently isolated in China from 1998 until 2013. These groups are 4bii f clade and 4bii d clade. Isolates in clade d span three continents from 1983 to 2001. Isolates in this clade have been inferred to originate from two isolates in 1995, one from a budgerigar in Turkey, the other from a pigeon in Germany [
34]. In this clade, Chinese, European and American isolates clustered on a separate branch from Middle Eastern isolates. Chinese PPMV-1 in this clade were isolated one or two decades ago, with the exception of pi/CH/SD2012, which was isolated in the Shandong province of China in 2012 [
37]. These results suggested that viruses in the 4bii d clade were not widespread in China in recent decade.
Our four PPMV-1 isolates and most strains isolated from China in recent decade were highly homologous to Belgian isolates from 1998 to 2011, all of which clustered in 4bii f. This clade contained European and Middle Eastern viruses isolated from pigeons between 1998 and 2011. Based on the phylogenetic analysis and isolation date, 4bii d was the predominant clade in China 20 years ago. The clade 4bii f viruses began to emerge in the late 2000s, when the clade 4bii d viruses began to decline.
PPMV-1 isolates in 4bii f mainly diverged into three branches, termed Belgium/98-like, P4-like and Belgium/11-like in this study. All three branches originated from PPMV-1 strains such as PPMV-1/Belgium/98–231/1998, PPMV-1/Belgium/98–238/1998 and PPMV-1/Belgium/98–248/1998 isolated in Belgium around 1998. ND132, ND163, ND167 and ND168 were classified into different branches of 4bii f. ND132 clustered with P4 and W4 on a separate branch of the 4bii f clade. Both P4 and W4 were isolated from wild birds in Guangdong province, south of China; P4 was from a wild pigeon in 2003 and W4 from a white-breasted water hen in 2005. Molecular dating based on different genes suggests that these viruses diverged from a common ancestor around 1999 [
39]. Viruses belonging to this branch were continually isolated in distinct provinces of China from 2003 until 2013, suggesting P4-like PPMV-1 has long been circulating throughout China.
The genomic sequences of three Shanghai NDV isolates, ND163, ND167 and ND168, showed the highest homology to PPMV-1/Belgium/11–08304/2011 (99.02%) [
42]. The nucleotide and amino acid identities of six viral proteins were even higher, at 99.04–100%. All other isolates in this branch were isolated in China since 2011. This result suggested that the viruses diverged from a common ancestor around 2011 and presumably were transmitted from Europe. In summary, our four isolates represented two groups of PPMV-1 prevailing in China in recent years. The P4-like group emerged around 1998 and the Belgium/11-like group around 2011.
Our cross-HI and cross-VN results showed antigenicity differences between the commonly used vaccine LaSota and current PPMV-1 strains in China, especially ND167, which is in a clade of the Belgium/11-like group. The R values of NDV167 compared with LaSota were 0.287 in cross-HI assays and 0.208 in cross-VN assays, suggesting that PPMV-1 strains prevailing in China were antigenically changing. The specific amino acid substitutions in the F and HN proteins of NDV167 may have led to major antigenicity differences, such as M14 T, P28S, V179I, A203S, V506I in F protein and I59V, A66V, L74I, T232 N, S432H, A497T, D569E in HN protein. Recent Chinese and European isolates of PPMV-1 diverged from a common origin and kept changing, probably due to immune pressure of the live NDV vaccines such as LaSota. These mutations led to antigenic variation and will probably result in the inefficiency of the vaccine in the foreseeable future.
Phylogenetic analysis of the entire genome of four PPMV-1 isolates in this study showed that the viruses prevailing in China in recent years originated from Europe, and had high identity with Belgian isolates from 1998 to 2011. No virulence-related mutations were found in the six viral genes of the current Chinese isolates compared to the Belgian isolates. To determine the virulence of those viruses, we measured pathogenicity to pigeons of ND132 in the P4-like group and ND167 in the Belgium/11-like group.
All pigeons infected with ND132 and ND167 showed transient clinical signs but no mortality when pigeons were infected with PPMV-1 via oral administration, intramuscular injection, or intravenous injection. In contrast, the chicken-origin virulent NDV strain F48E8 caused 100% morbidity; three pigeons died and four survived. The genotype VIb PPMV-1 appeared to be less pathogenic to pigeons than the chicken-origin strain F48E8 did, which was consistent with the ICPI values. In previous studies, the pathogenicity of VIb to chickens was determined to be mesogenic [
37,
38]. Thus, the 4bii PPMV-1 strains used in this study displayed the same pathogenicity as reported for European isolates [
43‐
46]; pigeons seemed to be resistant to those viruses. In other words, no definitive evidence confirmed a marked increase in the pathogenicity of recent Chinese isolates to pigeons. The high mortality clinically observed in China may not be caused simply by genetic variation of endemic strains; the influence of the pigeon lineage, co-infection with other pathogenic microorganisms, feeding environment, and even local climate need to be reconsidered.
A notable problem is viral shedding from infected pigeons. Our results showed that infected pigeons continued to shed, even at 3 weeks after infection, suggesting healthy pigeons could be PPMV-1 carriers. Based on epidemiological surveys, genotype VIb strains were identified from healthy-looking pigeons and doves. All isolates had a virulence motif of
112RRQKRF
117 in the F protein [
3,
47]. The virulent isolates from symptomless pigeons from 2011 to 2013 in live poultry markets clustered in the 4bii f clade [
3] and were highly homologous to our isolates. We propose that recent 4bii velogenic PPMV-1 isolates tended to develop a symbiotic relationship with pigeons so that infected pigeons showed no symptoms, became healthy carriers and shed occasionally. Previous studies showed that the same PPMV-1 strains were equally pathogenic to chickens and pigeons [
48,
49]. The disease in pigeons occurs because of spread from diseased chicken flocks and from domesticated or feral pigeons to poultry [
50]. Pigeon-origin subtype VIb viruses are widely considered to be responsible for the third pandemic of the 1980s. As reported in this study, current PPMV-1 isolates are gradually changing and giving rise to antigenic differences from vaccine strain LaSota, suggesting that PPMV-1 will be an imminent threat to NDV control worldwide.