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Characterization of Newcastle disease virus obtained from toco toucan

Given that the current Newcastle disease virus (NDV) infection in wild birds poses the threat to poultry, surveillance of Newcastle disease in captive wild birds was carried out in Jilin, China in 2018. Here, an NDV strain obtained from toco toucan was firstly characterized. The results showed that the F gene of the NDV isolate Toucan/China/3/2018 is classified as genotype II in class II. Sequence analysis of the F0 cleavage site was 113RQGR/L117, which supports the result of the intracerebral pathogenicity index assay indicating classification of the isolate as low-pathogenicity. Experimental infection demonstrated that Toucan/China/3/2018 can effectively replicate and transmit among chickens. To our knowledge, this is the first report on genetically and pathogenically characterizing NDV strain isolated from toucan, which enriches the epidemiological information of NDV in wild birds.


INTRODUCTION
Newcastle disease (ND) of poultry or ND virus (NDV) infection of wild birds is regarded as a highly contagious disease [1]. It is caused by NDV that is classified as a member of the genus Othoravulavirus within a new subfamily Avulavirinae of the family Paramyxoviridae [2]. In the evolutionary history of the past nearly 100 years, NDV strains are unremittingly evolving with numerous new genotypes and sub-genotypes emerging. Based on the analysis of the full F gene sequence, NDVs are classified into two major groups: class I and class II. Class I contains 1 genotype and class II includes 21 genotypes (I-XXI) [3].
NDV has a wide host range, with at least 250 species in 27 of the 50 orders of birds reported to be capable of natural or experimental infection by NDV [4,5]. Based on the whole information of NDV sequences registered in GenBank from 1930 to April 11, 2019, there are 1,425 strains naturally isolated from at least 169 species of wild birds (data not shown). Among the 17 orders,the most isolates were detected among wild birds in the order Anseriformes (n = 649), followed in frequency by the order Columbiformes (n = 153), Galliformes (n = 139), and Pelecaniformes (n = 135) (data not shown). It raises concerns regarding the potential role of wild birds in transmission of NDV. However, epidemiological information about NDV strains circulating in wild birds is still extremely limited.

Virus isolation and identification
The initial isolation of NDV was performed in 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs. The allantoic fluid samples were tested using hemagglutination and hemagglutination inhibition assays with standard NDV-specific sera according to the World Organisation for Animal Health (OIE) standard [7]. The NDV isolate was further purified in primary chicken embryo fibroblast cells by plaque assay and then propagated using SPF embryonated chicken eggs. The viral full-length genome sequence was amplified by reverse transcription polymerase chain reaction and sequencing with 11 pairs of primers provided in Table 1.

Sequence alignment and construction of phylogenetic tree
Assembly of sequences, translation of nucleotide sequences into amino acid sequences, and initial multiple sequence alignments were performed with the Clustal V method using MegAlign software version 1.03 (DNAStar, USA). Phylogenetic tree from the nucleotide sequences of the full-length F-genes obtained from the GenBank database was constructed based on 1,000 bootstrap replicates using the maximum-likelihood (ML) approach implemented in MEGA 7.0 software with 1,000 iterations [8]. Genetic distances based on the ML phylogenetic tree were calculated applying Kimura's two-parameter method.

Pathogenicity assessment
To evaluate the pathogenic potential of NDV obtained from toco toucan to chickens, two approaches were used. One is an in vivo pathogenicity test to determine the intracerebral pathogenicity index (ICPI) in 1-day-old SPF chicks according to the OIE standard procedures [7]. The other is a molecular pathogenicity approach to analyze the amino acid sequence of the F0 cleavage site.

Cohabitation infection
Two-week-old SPF White Leghorn chickens (n = 18) purchased from Weike Biotechnology(China) were randomly divided into three groups. Group 1 contained 12 chickens, of which 6 chickens were inoculated intranasally with 0.1 mL of 10 6.0 EID 50 /0.1 mL of allantoic fluid and another 6 chickens were raised together with them for cohabitation infection. Group 2 with 3 chickens was intranasally inoculated with PBS as a negative control. Group 3 with 3 chickens was intranasally inoculated with 0.1 mL of 10 6.0 EID 50 /0.1 mL of allantoic fluid to be used to analyze the tissues tropism of the virus. All chickens were monitored daily for clinical signs of disease and mortality during the 14day period. Oropharyngeal and cloacal swabs from each chicken every day were collected and inoculated into the allantoic fluid of 10-day-old SPF embryonated chicken eggs to determine the shedding or transmission potential of the virus. The virus titer was tested using hemagglutination assay according to the OIE standard [7]. The tissue samples from 3 chickens in group 3 at 7 days post inoculation (dpi) were collected and fixed by immersion in 10% neutral buffered formalin for approximately 24 h, then 3 µm sections were prepared for immunohistochemistry (IHC) as previously described [9].

Calculation of antigenic similarity
The antigenic difference between the NDV isolate obtained from toco toucan and vaccine strain LaSota was antigenically analyzed by cross-HI test using the chicken antisera against Toucan/China/3/2018 and LaSota from 14 dpi. The serological relationship was determined by calculating the coefficient of antigenic similarity (R) between each pair of strains. The formula was used: , where γ1 is the ratio of titer of strain A with antiserum B and titer of strain A with antiserum A; γ2 is the ratio of titer of strain B with antiserum A and titer of strain B with antiserum B. When R = 100%, antigenic relatedness is 100%; if R = 50%, antigenic relatedness is 50%; if R = 25%, antigenic relatedness is 25%, etc. In general, R ≤ 25% indicates significant antigenic difference [10].

Ethics statement
This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People's Republic of China. The protocols for animal studies were approved by the Committee on the Ethics of Animal Experiments of Jilin University (approval numbers 2018051377-5 for chicken eggs and 2018071599-3 for chickens).

Genomic characteristics
One NDV strain from 1 of 4 toucans in this study was named as Toucan/China/3/2018, the nucleotide sequence of which is available in GenBank under accession number MK902652. The genomic characteristics of Toucan/China/3/2018 were shown in Table 2. The lengths of 3′ leader and 5′ trailer are 55 and 114 nt, and the 5′ untranslated regions (UTRs) of six genes are always longer than 3′ UTRs. The gene start sequence of L gene is ACGGGTAGGAC, while that of the other five genes is ACGGGTAGAA. The gene end sequence for NP, M, and L genes is TTAGAAAAAA, while for that of P and HN genes is TAAGAAAAAA, and for that F gene is TTAGAAAAAA. The lengths of intergenic sequence (IGS) of P-M and M-F are 1 nt, while the IGS of NP-P, F-HN, and HN-L are 2 nt, 31 nt, and 47 nt, respectively.

Phylogenetic analysis
Based on the phylogeny of the open reading frame (ORF; 1-1,662 nt) of the F gene, the phylogenetic tree of at least 20 distinct genotypes (I-XXI, except for genotype XV that contains only recombinant sequences) showed that Toucan/China/3/2018 is classified as genotype II (Fig. 1). Further analysis displayed that Toucan/China/3/2018 with those strains mainly isolated from pigeons and chickens clustered into a separated clade, which formed a sister relationship with the other cluster where the used vaccine strains, LaSota, Clone 30, B1, and VG/GA, during the past 60 years (Fig. 2).

Pathogenicity evaluation
The pathotype of Toucan/China/3/2018 was assessed by two approaches: an in vivo pathogenicity test (ICPI assay) and a molecular pathogenicity approach (analysis of amino acid sequence of the F0 cleavage site). The result of the ICPI assay indicated that Toucan/ China/3/2018 had an ICPI value of 0.6, which is below the OIE value of equal to or greater than the 0.7 standard for virulent NDV. Thus, the isolate was classified as low-pathogenicity.
Sequence analysis of the F protein gene has been used as an important molecular tool to predict the pathotype of NDV isolates [7]. In the present study, the ORF of the F gene of the isolate Toucan/China/3/2018 contained 1,662 nucleotides, encoding for 553 amino acids. The deduced amino acid sequence of this gene at the cleavage site was 113 RQGR/L 117 , which supports the result of the ICPI assay indicating classification of the isolate as low-pathogenicity.

Transmission potential
To investigate the potential for horizontal transmission of Toucan/China/3/2018, 6 SPF chickens were inoculated intranasally with the virus and another 6 chickens were housed 4/10 https://vetsci.org https://doi.org/10.4142/jvs.2020.21.e19 with them. During the 14-day observation period, the chickens by intranasal inoculation and chickens for cohabitation infection did not show any obvious clinical signs, and none of these chickens died. The supernatant of oropharyngeal and cloacal swabs was inoculated into the allantoic fluid of 10-day-old SPF embryonated chicken eggs. Within 72 h of incubation, no chicken embryos died. After that, allantoic fluid was collected and detected its hemagglutination activity. The result showed that the virus could be detected in swabs from 2 to 14 dpi for the 6 chickens after intranasal inoculation in group 1, while the virus shedding was detected in 1 to 6 of 6 naïve-contact chickens from 4 to 14 dpi in group 1 ( Table 3). Fig. 3 showed that the chicken by intranasal inoculation at 7 dpi was positive for NDV antigen in almost all tissues and organs such as brain, trachea, lung, heart, liver, spleen, glandular stomach, intestine, pancreas, kidney, thymus, and bursa of Fabricius, indicating that Toucan/China/3/2018 has a broad tissue tropism. showed Toucan/China/3/2018 had only 1 mutation in the heptad repeat C region, and only 1 mutation in the major transmembrane domain, when compared with the consensus amino acid sequence derived from LaSota ( Table 4). As for the HN comparison, no amino acid substitution was observed at the antigenic sites between them ( Characterization of toucan NDV  The antigenic relationship was determined by the cross-HI test ( Table 6). Using the R value as described above, it was found an R = 70.71% value, demonstrating a definite difference in antigenicity between Toucan/China/3/2018 and LaSota. This finding was in agreement with the result of phylogram F of genotype II (Fig. 2).

DISCUSSION
Studies on the diversity, distribution, and potential impact on the poultry industry of NDV circulating in wild birds are extremely limited. As a kind of active and colorful bird, toucans are mainly distributed in tropical forests of South America and become on the verge of extinction. Historically, NDV was isolated from three of 48 toucans in a quarantine station in the United States in the early 1970s [6]. But its genetic background is unclear because of no sequence to follow. In this study, an NDV strain was obtained from toco toucan in China in 2018. To our knowledge, this is the first report on genetically and pathogenically characterizing NDV strain isolated from toucan and its sequence had been registered in GenBank for the first time.
The transmission of NDV occurs through inhalation or ingestion of the virus in aerosol, respiratory discharge and feces from infected birds or contaminated feed, water, equipment and clothing [11]. Animal experiments in this study showed that the virus can be detected in 1 to 6 of 6 chickens for cohabitation infection from 4 to 14 dpi, with the highest rate of virus detection at 8 dpi ( Table 3). It thus indicated that Toucan/China/3/2018 can transmit between chickens by naïve contact. However, it is unclear how NDV was introduced to toucan. As we know, these investigated toucans had no vaccination history in order to avoid stress. It is necessary to explore whether a vaccine strain spills over from other birds to toucan. As shown in Fig. 2, although Toucan/China/3/2018 clustered into a cluster different from that of the common vaccine viruses in the phylogenetic tree of genotype II, it had a close relationship with Pigeon/China/101/08. Thus, it is speculated that the virus is more likely to originate from other wild birds such as pigeons. Characterization of toucan NDV -Dots indicate residues identical to LaSota.