Molecular epidemiology of Newcastle disease virus isolates from vaccinated commercial poultry farms in non-epidemic areas of Japan

All suspected NDV isolates yielded a 766-bp product in the nested PCR amplification step. This confirmed that all isolates belong to Avian Paramyxovirus type-1 viruses. All strains exhibited mean death time (MDT) of 48 to 56 hours in embryonated chicken eggs. Intracerebral pathogenicity index (ICPI) values ranged from 1.7 to 1.9 (Table 1). Nucleotide sequence analyses of the variable region of the F-gene (47–421 nt) showed that all isolates had multiple basic amino acids at its F0 proteolytic cleavage site (residues 112–117). Predicted amino acid sequence of the F0 cleavage site was ¹¹²RRQKR¹¹⁶ at the F2 protein and phenylalanine (¹¹⁶ F¹¹⁷) at the N-terminus of the F1 protein for all strains. These results indicated that all isolates were velogenic.

A total of 1662 nucleotides encoding for 553 amino acid residues were identified in the complete coding region of the F-gene of all field strains. Six potential N-glycosylation sites (Asn-X-Ser/Thr where X is any amino acid except proline or aspartic acid) located at positions 85 to 87, 191 to 193, 366 to 368, 447 to 449, 471 to 473 and 541 to 543 were recognized. Twelve cysteine residues located at positions 25, 76, 199, 338, 347, 362, 370, 394, 399, 401, 424 and 523 were identified. Comparison of glycosylation sites and cysteine residues showed no changes in the amino acid sequence in all field strains, which may indicate that these sites were highly conserved.

Analysis of seven neutralizing epitopes located at positions 72, 74, 75, 78, 79, 157 to 171 and 343 of the F-protein showed a K to R amino acid substitution at position 78 in seven of the nine strains (Table 2). These strains were JP/Ibaraki/SG106/99, JP/Ibaraki/IS5/02, JP/Chiba/BY103/01, JP/Ibaraki/IS2/02, JP/Miyagi/AGT/02, JP/Chiba/BY7/02 and JP/Fukushima/NYF-3/02. Furthermore, analysis of amino acid substitutions showed 13 point mutations in the variable region of the F-gene of the Japanese field strains (Table 2). Genotypic and subgenotypic-specific amino acid substitutions were also observed, which were consistent with the proposed theory of NDV evolution as reported previously [18].

Phylogenetic analyses of nine field strains and 180 NDV strains from GenBank were performed by using contiguous nucleotide sequences of the F-gene, NP-gene and L-gene. Reference strains from GenBank were selected as representatives of nine of the 11 ND genotypes (genotypes I to XI) representing isolates from different regions of the world (Figures 1, 2 and 3).

Field NDV strains were observed to belong to two distinct genotypic groups (genotype VI and VII) using phylogenetic analysis of the complete coding sequence of the F-gene. Early isolates such as JP/Osaka/2440/69 and JP/Ibaraki/SM87/87 were genotype VI, while field strains isolated from 1999 onwards were from genotype VII (Figures 1 and 4). Phylogenetic analysis using the 3-prime portion of NP-gene and 5-prime portion of L-gene yielded the same tree topology and phylogenetic groupings (Figures 2 and 3). Phylogenetic analyses at the subgenotype level using the variable region of the F-gene sequences revealed that JP/Osaka/2440/69 belongs to subgenotype VIa, JP/Ibaraki/SM87/87 to subgenotype VId and all the recent field isolates to subgenotype VIId (Figures 5 and 6).

F-gene nucleotide sequence of JP/Osaka/2440/69 was found to be closely related (98.5-99.2% nucleotide sequence identity) to isolates from the Middle East. The F-gene sequence of JP/Ibaraki/SM87/87 was closely related (96.3-100.0%) with isolates from Japan and China. All the other VIId isolates were highly similar (97.2-100.0%) to isolates from Japan, China and Taiwan and interestingly to a goose isolate from China (98.4-98.6%) (Table 3).

No intragenic nor intergenic recombination events (Unique events = 0; Recombination signals = 0) were observed involving the field isolates using all the described methods. Analysis of the over-all mean evolutionary distance among the Japanese field isolates showed rates of 3.9×10^-2 [standard error (s.e) 0.01], 3.1×10^-2 (s.e. 0.01) and 2.0×10^-2 (s.e. 0.01) base substitution per site in the full F-gene, partial NP-gene and partial L-genes, respectively. In contrast, over-all mean evolutionary distance among the recent field strains (1999–2002) were 1.0×10^-3 (s.e. 0.001) base substitution per site for the F and NP-genes and zero base substitution for the L-gene. In addition, 25 sites in the F-gene of all field strains were observed to be under negative selection (p-value < 0.05) (Table 4).

Nine NDV strains isolated from commercial poultry farms with different spatial (Osaka, Ibaraki, Chiba, Fukushima and Miyagi Prefectures) and temporal distribution patterns (1969, 1987, 1999, 2001 and 2002) were used to investigate the molecular epidemiological relationships of ND outbreaks in vaccinated commercial poultry flocks in Japan. These strains were isolated by one to two passages of pooled infected tissues in 10-day-old embryonated specific pathogen-free (SPF) chicken eggs. Infective allantoic fluids were harvested and kept in lyophilized form or in serum tubes and stored at −80°C until further use. All isolates used in this study were provided by Poultry Products Quality Control Co. Ltd. (Fukushima, Japan).

Records of management and farm history were obtained to characterize the clinical profile of nine suspected NDV strains (Table 1). The oldest strain was from Osaka Prefecture in 1969 (JP/Osaka/2440/69). This isolate was recovered from six dead layer birds that were submitted to the Osaka Veterinary Municipal Office for diagnosis. The flock was vaccinated with the live B1 vaccine in drinking water at 5–7 days of age and killed ND vaccine (Sato strain) at 25 days. The affected flock was around 80 days of age when the disease occurred. Mortality was reported to be around 60-70% but no data were given regarding observed clinical signs and production performance. The second strain was from a layer farm in Ibaraki Prefecture isolated in 1987 (JP/Ibaraki/SM87/87). Total population of affected farm was 33,360 birds in 12 open-type houses of 2,780 birds each. The disease was reported in one of the houses. The flock was vaccinated with the live B1 spray at 10 days of age, killed Ishii/B1 at 45 days, live B1 spray again at 60 days and killed Ishii/B1 again at 120 days. The disease occurred two months after the last vaccination at 180 days of age. The disease was characterized by gasping and 25% drop in egg production. Mortality was less than three percent. The third strain was from Ibaraki Prefecture in 1999 that was isolated from dead spent hens sent for diagnosis (JP/Ibaraki/SG106/99). The isolate was from an unknown farm raising spent hens for liquid egg production. No detailed farm information was obtained. Chickens submitted for diagnosis were approximately more than 700 days old. Necropsy findings were proventriculitis, hemorrhagic lesions in duodenum and petechiae in lymphocytic tissues. The fourth strain was from a replacement pullet farm in Chiba Prefecture isolated in 2001 (JP/Chiba/BY103/01). The disease occurred in a flock of 21,000 birds. The flock was vaccinated with the live B1 strain in drinking water at 4 and 10 days of age, live B1 spray at 28 days and killed Ishii strain at 45 and 90 days of age. It was reported that the killed vaccines were injected by hired professional vaccination staff that travel from farm to farms. Six days after last vaccination, the disease occurred characterized by gasping, nervous symptoms, leg weakness, twisting of neck and greenish diarrhea. Mortality was around 10%. The fifth strain was from a layer farm in Ibaraki prefecture isolated in 2002 (JP/Chiba/BY7/02). The affected farm had a population of 125,000 birds (five houses of 25,000 birds) and affected flock was around 25,000 birds (one house). The flock was vaccinated with the live B1 strain in drinking water at 10 days of age, live B1 spray at 24 days, killed Ishii strain at 45 days, live B1 spray again at 60 days and killed Ishii strain again at 95 days. The disease occurred at 110 days of age characterized by gasping, swollen face and infectious bronchitis (IB)-like respiratory signs and 20% drop in egg production without significant mortalities. Necropsy findings were necrotic ovarian follicles and necrotic catarrhal inflammation of the intestines. The sixth strain was from a layer farm located in Ibaraki prefecture in 2002 (JP/Ibaraki/IS5/02). The affected farm had a population of approximately 200,000 birds and the affected flock was around 41,000 birds. The flock was vaccinated with the live B1 spray at 10 and 28 days old, killed Ishii strain at 45 days, live B1 spray again at 60 days and killed Ishii strain again at 90 days. The disease occurred at 336 days of age characterized by mild respiratory signs such as gasping, seven percent decrease in egg production with no marked mortalities. The seventh strain was recovered from dead birds from a replacement pullet farm in Ibaraki prefecture in 2002 (JP/Ibaraki/IS2/02) that were submitted for diagnosis. Total farm population was around 10,000 birds. The flock was vaccinated with the live B1 spray at 10 days of age. The disease occurred four days after vaccination characterized by severe depression. The eight strain was isolated from a layer farm in Fukushima prefecture (JP/Fukushima/NYF-3/02). The farm had a population of around 120,000 birds and affected flock was approximately 16,000 layers. The flock was vaccinated with the live B1 spray at 10 and 28 days of age, killed Ishii strain at 45 days, live B1 spray again at 60 days and killed Ishii strain again at 90 days. The disease occurred around 532 days of age characterized by mild gasping, increase in soft-shelled eggs and eight percent decrease in egg production. Mortalities were minimal and within production standards. The ninth strain was isolated from a layer farm in Miyagi prefecture in 2002 (JP/Miyagi/AGT/02). The flock was vaccinated with the live B1 in drinking water at 10 days of age, live B1 spray at 24 days, killed Ishii strain at 45 days, live B1 spray again at 60 days and killed Ishii strain at 95 days. It was informed that the flock was kept in multiple age houses (six different flocks in one house). Disease occurred at 250 days of age characterized by gasping, greenish white diarrhea and 70% decrease in egg production. No marked mortalities were observed.

Biological and pathotypical characterization of isolates were performed using mean death time (MDT) in 10-days-old embryonated SPF chicken eggs and intracerebral pathogenicity index (ICPI) in 1-day old chicks according to the protocols described in the OIE manual [34]. Confirmation of pathotypes was performed by nucleotide sequence analysis of the F0 proteolytic cleavage site (residues 112–117). Pathotypical data for strain JP/Fukushima/NYF3/02 were obtained in our previous study.

Nested RT-PCR was performed to confirm the identities of suspected NDV strains. In brief, isolates were propagated once in 10-day-old embryonated SPF eggs. Viral RNA from infected allantoic fluids was extracted directly by using a QIAamp® Viral RNA Mini Kit (Qiagen, West Sussex, UK). Viral RNA was transcribed to cDNA by using random hexamers and Primescript® Reverse Transcriptase (Takara Bio-Inc, Shiga, Japan). cDNA was amplified by PCR as described previously [16]. A two-step nested PCR was performed to amplify the region comprising the 3′ end of the M-gene and the 5′ end of the F-gene using KOD dash® (Toyobo, Osaka, Japan), 5 uM of external and internal primers as described by Mase et al.[16] (Table 5). Thermocycling conditions for the first and second PCR steps were as follows: prewarming at 94°C for 2 min (1 cycle), denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec and extension at 72°C for 1 min. Amplification steps were performed for 35 cycles. The final extension was performed at 72°C for 30 sec (1 cycle).

Confirmed NDV strains were subjected to additional RT-PCR amplification to characterize the open reading frame of the F-gene sequences (4504–6295 nt of whole NDV genome), 3-prime end of the NP-gene sequences (16–783 nt of whole NDV genome) and 5-prime end of the L-gene sequences (13995–14719 nt of whole NDV genome). In brief, RT-PCR was performed by using SapphireAmp® Fast PCR Master Mix (Takara Bio), 5 uM of forward and reverse primers (Table 5) and cDNAs that were transcribed previously. Thermocycling conditions consisted of initial denaturation at 95°C for 2 min followed by 35 cycles of 98°C for 10 sec (denaturation), 55°C for 10 sec (annealing), 72°C for 10 sec (extensions) and final extension at 72°C for 2 min. PCR products were analyzed by electrophoresis with 1.2% agarose gel and purified by using QIAquick® Gel Extraction Kit (Qiagen, Valencia, CA). The nucleotide sequences of PCR products were determined by Big Dye terminator cycle-sequencing kit version 3.1 (Applied Biosystems Inc., Foster City, CA) and an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). DNA products were sequenced from both directions. The complete F-gene sequence for strain JP/Fukushima/NYF3/02 was obtained in our previous study.

Sequence assembly and editing were performed using CodonCode Aligner® (version 3.7.1, CodonCode Corporation, MA) and ClustalX® (version 2.1, Conway Institute UCD Dublin, Ireland). Deduced amino acid sequences were determined using Bioedit® software package version 7.1.3.0 [35]. Confirmation of identity and homology were performed using BLAST http://​www.​ncbi.​nlm.​nih.​gov.

To determine the molecular epidemiological relationships of field strains, 180 NDV strains isolated from different regions of the world at different time periods were obtained from GenBank. These reference strains were representatives of all the different NDV genotypes and subgenotypes. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4 [36]. Phylogenetic trees of the variable region of the F-gene sequences (47–421 nt), complete coding region of the F-gene sequences (1–1662 nt), coding region of the 3-prime end of NP-gene sequences (1–622 nt) and coding region of the 5-prime end of L-gene sequences (5629–6333 nt) were constructed by the neighbor-joining method with the maximum composite likelihood substitution model at 1000 bootstrap replicates.

Intragenic recombination events in the NDV nucleotide sequences were determined using RDP v3.44 program. [37]. Seven different algorithms integrated in the program namely RDP, GeneConv, Bootscan, MaxChi, Chimaera, SiScan and 3Seq were applied to detect any putative recombination breakpoints and to estimate the occurrence of any recombination events within all the analyzed genes. Sequences with recombination events identified by at least two detection methods (p < 0.01) were considered as true recombinants. Intragenic recombination events within the F-gene were also determined by comparing the tree topology of phylogenetic analyses using the variable region (47–421 nt) and complete coding region (1–1662 nt) of the F-gene. Intergenic recombination events were determined by comparison of topologies of the generated F, NP and L-gene phylogenetic trees.

Evolutionary distances were calculated using MEGA version 4 using the Maximum Composite Likelihood method [36]. Codon positions included were the 1st + 2nd + 3rd + noncoding. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). Estimates of s.e. were obtained by a bootstrap procedure of 1000 replicates. Analysis of evolutionary selection profile was performed using Datamonkey http://​www.​datamonkey.​org/​ following the Fixed- Effect Likelihood (FEL) method [Hasegawa, Kishino and Yano (HKY) model, p-value less than 0.05] [38, 39].

Nucleotide sequences used in this study were submitted to the DNA Databse of Japan (DDBJ) with the following accession numbers: JP/Osaka/2440/1969 F-gene sequence: AB853926; NP-gene: AB854728; L-gene: AB854729. JP/Ibaraki/SM87/1987 F-gene sequence: AB853928; NP-gene: AB854730; L-gene: AB854731. JP/Ibaraki/SG106/1999 F-gene sequence: AB853927; NP-gene: AB854732; L-gene: AB854733. JP/Ibaraki/BY103/2001 F-gene sequence: AB853931; NP-gene: AB854734; L-gene: AB854735. JP/Ibaraki/BY7/2002 F-gene sequence: AB853930; NP-gene: AB854736; L-gene: AB854737; JP/Ibaraki/IS5/2002 F-gene sequence: AB853933; NP-gene: AB854744; L-gene: AB854745. JP/Ibaraki/IS2/2002 F-gene sequence: AB853932; NP-gene: AB854742; L-gene: AB854743; JP/Fukushima/NYF-3/2002 F-gene sequence: AB853329; NP-gene: AB854740; L-gene: AB854741; JP/Miyagi/AGT/2002 F-gene sequence: AB853929; NP-gene: AB854738; L-gene: AB854739.

Animal experiments in this manuscript were critically reviewed and approved by the Animal Ethical Committee of Poultry Products Quality Control (Permit number: 2013-03A) and performed in accordance with the Japanese laws and international guidelines for the use of animals in research.