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A Genotype Newcastle Disease Virus Isolated from Infected Chicken Flocks in ChinaWanying Qu1,#, Xiaohua Wang2,#, Gang Chen1, Ying Jing1, Jinfeng Gao1, Kai Zhao1,3,*

1Key Laboratory of Microbiology, School of Life Science, Heilongjiang University, Harbin 150080, People’s Repub-lic of China2Heilongjiang University Library, Heilongjiang University, Harbin 150080, People’s Republic of China3School of Biological Science and Technology, University of Jinan, Jinan 250022, People’s Republic of China

Case Report Open Access

Journal ofVirology and Vaccines Research

Received Date: September 10, 2017 Accepted Date: October 08, 2017 Published Date: October 10, 2017

Citation: Kai Zhao (2017) A Genotype Newcastle Disease Virus Isolated from Infected Chicken Flocks in China. J Virol Vaccines Res 1: 1-9.

*Corresponding author: Kai Zhao, KeyLaboratory of Microbiology, School of Life Science, Heilongjiang University, Harbin 150080, People’s Republic of China; Tel: +86 451 86608586; Fax: +86 451 86609016; E-mail: zybin395@126.com #These authors contributed equally to this work

©2017 The Authors. Published by the Clerisy under the terms of the Crea-tive Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited.

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Introduction Newcastle disease (ND) is a highly infectious dis-ease, which causes devastating losses in both commercial and home-kept chicken chickens. The causative agent of the ND is designated as Avian paramyxovirus serotype 1, which is iden-tified as a negative-sense, single stranded, non-segmented RNA virus, it also belongs to the genus Avulavirus within the family Paramyxoviridae [1]. Newcastle Disease Virus (NDV) is classified into class I and class II [2,3]. Class I with the ge-nome size of 15,198nt is accidentally isolated from the wild aquatic birds and domestic fowls, which normally is aviru-lent; Class II exists primarily in the wild and domestic poul-try, which includes the most poisonous and some avirulent NDVs.

For example, the genotypes I-IV viruses with the genome size of 15,186nt are early lineage before 1960, whereas the geno-types V with the genome size of 15,192nt are recent lineage after 1960 [4,5]. According to isolate time and genetic dis-tance, the genotype VI and VII are further divided into eight subgenotypes [5]. Furthermore, the genotypes VI and VII of class II are the most popular and widespread in the world [6]. All NDV strains are assigned to a single sterotype, but inactivated vaccines and attenuated live vaccines still can-not effectively prevent ND outbreak due to genetic variation of NDV epidemic strains [7], and these NDVs exhibited high genetic diversity and multiple genotypes [8]. However, the genotype VII NDV has been the main pathogen of ND oc-currence since 1990, and according to previous reports [9], the genotype VII NDV has become a major epidemic virus in the past 10 years. The virus is related to the recent outbreaks of Newcastle disease in Asia, Europe, Africa, Middle East and South America [10,11].

Abstract ND is a highly infectious disease, which causes massive losses in both commercial and home-kept chicken. A Newcastle Disease Virus (NDV) isolate was collected from the infected chicken flocks during the outbreaks of NDV in Dehui city, Jilin province, China in 2010. The isolate, named as strain JL-DH, was analyzed by using Intracerebral Pathogenicity Index (ICPI), and the genotype was characterized by RT-PCR. The strain JL-DH was identified as a genotype. NDV by the serological detec-tion and sequence alignment assay, and the genotype is predominantly responsible for ND outbreaks in China. The strain JL-DH possessed the ICPI value of 1.86, which showed it was highly lentogenic. And the amino acid cleavage site of strain JL-DH was 112R-R-Q-K-R-F117. The study provided one candidate strain for the research and development of NDV vaccine in future. Keywords: Newcastle disease virus; Isolation; F gene; Biological characterization; Genotype

It was reported that the genotype VII NDV reduced the egg production of chickens and had a bad effect on the chickens’ respiratory system and nervous system [12]. Because the clin-ical symptoms of ND are highly variable, and no pathogno-monic signs can be observed, the absolute diagnosis mainly depends on the isolation and identification of the causative virus [13,14]. In this study, one Newcastle disease virus strain JL-DH from the NDV-infected chickens in Dehui city, Jilin province of China was isolated, and the strain JL-DH was a genotype NDV, which can effectively prevent epidemics, oc-curs and solve the problem of declining egg production. Ad-ditionally, a candidate strain for the research and develop-ment of NDV vaccine in future was provided in this study.

Materials and MethodsEthics Statement Care of laboratory animals and experimentation on animals were done in accordance with animal ethics guide-lines and approved protocols. All the animal studies were approved by the Animal Ethics Committee of the Harbin Veterinary Research Institute of the Chinese Academy of Ag-ricultural Sciences (CAAS), China (SCXK (H) (2013-001).

Samples and Virus Isolation Samples of trachea, liver and duodenum of the in-fected chickens from chicken farms were obtained from De-hui City of Jilin Province in China during 2010. All of the samples were diluted in Phosphate-Buffered Saline (PBS) containing penicillin and streptomycin, then were vaccinated into the allantoic cavity of 10-day-old SPF eggs. The allantoic fluids were collected when the embryos passed away or 72 h through incubation at 37°C, subsequently, the allantoic flu-ids tested by Hemagglutination (HA) assays. Simultaneously, HA-positive samples were tested by Hemagglutination Inhi-bition (HI) assays [15].

Culture of Primary Chicken Embryo Fibroblast Cells Nine-day-old chicken embryos were provided by Harbin Pharmaceutical Group Bio-vaccine Co. Ltd, and the chicken embryos were placed in sterile petri dish under sterile conditions, afterwards, the head, claws, fins and viscera of the chicken embryos were removed, then the chicken embryos were washed with Hank’s solution and treated with trypsin to obtain a mono-disperse cell suspension. The cells suspension were placed in a 6-well culture plate and incubated at 5% CO2 and 37°C for 24h. Subsequently, allantoic fluid was diluted at 1:10,000 with 1×DMEM medium which was no serum and inoculated into abovementioned monolayer cells in a 5% CO2 incubator at 37°C. The Cytopathic Effect (CPE) were ob-served and recorded daily. When the CPE reached 70%, the HA titers were measured after three freeze-thaw cycles.

Plaque Purification of Newcastle Disease Virus Isolates For NDV plaque purification, chicken embryos cells were seeded in 6 cm petri dishes and washed thrice with PBS. The 0.5 ml of cells solution was poured into the dishes, and the infected cultures were incubated for 1h at 37°C within ac-cidental agitation. The dishes were incubated at 5% CO2 and 37°C, and plaques were observed after 48-72h by addition of covering (100μl) containing neutral red and incubated at 37°C prior to accomplishing the plaque purification [16].

Proliferation of Newcastle Disease Virus Isolate 0.2 ml of allantoic fluid including NDV was inocu-lated into the allantoic cavity of 10-day-old chicken embryo for virus proliferation. After culture at 37°C for 72h, the al-lantoic fluid was obtained, and tested by HA and HI assays according to the previous method [15].

Pathogenicity The Intracerebral Pathogenicity Index (ICPI) for the isolates was determined in line with Standard provisions. Briefly, one-day-old SPF chickens were inoculated intracer-ebrally within 50μl of filtered allantoic fluid from chicken em-bryos, which was diluted at 1:10. Chickens were monitored daily for 8 days, finally the scores for each virus were counted in a range from 0 to 2 [17]. The median embryo lethal dose (EID50) is carried out in the light of the Reed and Muench method [18].

Reverse Transcriptase-Polymerase Chain Reac-tion To perform the RT-PCR, the viral RNA was ex-tracted from infectious allantoic fluid and followed by am-plifing the DNA fragment to generate the F protein gene. A pair of oligonucleotide primers was designed according to the nucleotide sequences of NDV F protein gene embodied in GenBank using primer premier 5.0 software. The primer was synthesized by Sangon Biotech (Shanghai) Co., Ltd. RT-PCR was enforced by using a One-Step RT-PCR kit in accordance with the instructions of manufacturer for use. The PCR prod-ucts were analyzed by electrophoresis on a 2% agarose gel. Then, the PCR products were recovered and purified by using Shanghai Huashun Pellet Recycling Kits. Shanghai Huashun column gum recovery kit was concatenated PMD18-T vector (TaKaRa, Dalian, China) [9]. Then, being transformed into E. coli DH5α cells (TaKaRa, Dalian, China). Sequencing was conducted by Shanghai Biological Engineering Co., Ltd.

Analysis of Nucleotide and Amino Acid Sequenc-es Sequence edit, prediction and analysis were accom-plished by MegAlign procedures in the lasergene package. So as to identify the homology of the isolates to other NDV vi-ruses, these complete NDV genome sequences were received from GenBank. Subsequently, phylogenetic trees were paint-ed with MegAlign. Nucleotide and amino acid homology was reckoned by DNA Star software.

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Statistical Analysis All the experimental results were expressed as mean values ± Standard Deviation (SD). One-factor analysis of variance was employed to evaluate the statistical differences among different groups with SPSS 19.0 software. The differ-ence between groups with P<0.05 was considered to be statis-tically significant.

ResultsVirus Isolation One NDV strain was isolated from NDV-infected chickens and named NDV/Chicken/JL-DH/2010. The strain JL-DH was isolated from the NDV-infected chickens depend-ing on their positive HA. The isolate was determined as NDV based on HI. These results showed that the HA antibody titers of strain JL-DH ranged from 27 to 210 with anti-NDV chicken serum at 72h after inoculation. The HI titre of the primary CEF cells varied between 27 and 29 and it was controlled by NDV and AIV reference antiserum, which certificated the ex-istence of the NDV virus. And the cells displayed a cytopathic effect after inoculation for 36 h (Figure 1), the number of vi-rus syncytia gradually became larger. EID50 of strain JL-DH was 10-6.17/0.1 ml. The strain JL-DH showed a very high ICPI of 1.86, being classified into a virulent-type NDV.

Analysis of PCR Products Pathotyping of strain JL-DH was executed by RT-PCR which abided by BamH enzymes (Figure 2). The speci-ficity of products from RT-PCR was shown by DNA fragment in size of 1993 bp from strain JL-DH. Figure 2 showed that re-striction digest of RT-PCR products by BamH generated two DNA fragments in size of 2692bp and 1993bp, respectively. The fragment size of pMD18-T vector is the same as the size of 2692bp generated by restriction enzyme. And 1993bp is consistent with the size of target fragment. These results in-dicated that the target fragment has been connected to the pMD18-T Vector.

Homology Analysis of Nucleotide Sequence and Amino Acid Sequence Encoded By the F Gene To further characterize strain JL-DH, amino acid and nucleotide sequences of strain JL-DH displayed a closer phylogenetic relationship to 27 reference strains. The percent-age of nucleotide sequence identity of F gene between strain JL-DH and HG-Beijing-2009 strain (accession number: FJ882015) was 99.5%, and that of amino acid sequence iden-tity was 99.5%. This result was similar to genotypes HLJ363 (accession number: FJ480784), SG-Liaoning-2009 (accession number: FJ882014) and Guangxi4-2000 (accession number: DQ485258), which share 97.6%, 97.5% and 95.1% nucleotide, 97.8%, 97.7% and 96.4% amino acid sequence homology.

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The homologies of nucleotide and amino acid se-quences were comparatively low between distinct genotypes. For genotype strains, the nucleotide and amino acid homolo-gy between the strain JL-DH and the La Sota strain (accession number: AF077761) were 82.1 % and 87.5 %, respectively. The nucleotide and amino acid sequence homologies between the strain JL-DH and Mukteswar were 84.6% and 89.5% respec-tively. Furthermore, the percentage of nucleotide and amino acid sequences between strain JL-DH and Italian were 86.6% and 90.8%. These results indicated that the strain JL-DH is more familiarly related to genotype isolates in the light of the nucleotide and amino acid sequences (Figure 3).

Analysis of Nucleotide Genetic and Amino Acid Evolution of NDV F Gene Strain JL-DH was in the same branch as HG-Bei-jing-2009, HLJ363, SG-Liaoning-2009, Guangxi4-2000 and GD-Qingdao122011. There are the closest genetic relation-ships between the strain JL-DH and HG-Beijing-2009 strain. Amino acid evolution of strain JL-DH and other strains were far. This is consistent with similarity analysis results of the F gene nucleotide (Figure 4). The complete genome sequence of strain JL-DH has been submitted to GenBank, and accession number is JN032760.

Figure 1: Culture of chicken embryo fibroblast cells. (A) un-vaccinated cells; (B-F) cells at 18, 24, 40, 48 and 60 h after inoculation, respectively.

Figure 2: RT-PCR products amplified from strain JL-DH following electrophoresis stained 1% agarose gel (A) and the recombinant plasmid identified by BamH restriction endonuclease (B). M represents DNA Marker DL2000; Lane 1-4 of Figure 2A represent RT-PCR product of F gene; Lane 1 and 2 of Figure 2B represent recombinant plasmid digestion by Bam H and DNA Marker DL 15000, respectively.

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Figure 3: Homologous analysis derived by using nucleotide and amino acid sequences from strain JL-DH and reference sequences from GenBank. (A) Homology among the nucleic acid sequences; (B) Homology among amino acid sequences.

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Figure 4: Phylogenetic tree of strain JL-DH and reference sequences from GenBank based on nucleotide and amino acid sequences from NDV F genes. (A) Phylogenetic tree based on the nucleotide of the gene F; (B) Phylogenetic tree based on sequences of the gene F.

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Discussions ND is a highly epidemical viral disease of poultry, which is characterized by gastrointestinal and respiratory symptoms associated with nervous disorders [19]. In this study, one velogenic isolate of NDV was isolated from contaminated chicken flocks. And the velogenic isolate is convinced by us-ing specific antiserum in a haemagglutination inhibition test. Currently, RT-PCR-based technique for the detection and sys-tematization (genotyping) of APMV-1 RNA in allantoic fluid is becoming increasingly common in diagnostic laboratories [20-22]. The F0 cleavage site sequence was determined by RT-PCR, afterwards, the amplified products were analyzed by restriction enzyme, probe hybridization or nucleotide sequencing [23]. Sequence analysis of the F protein cleavage site is accustomed to predict prospective pathogenicity of NDV despite traditional methods such as Mean Death Time (MDT) and ICPI [24,25]. According to the regulation of the OIE, the virulence of NDV is connected with ICPI and the cleavage site in the F protein [26,27]. F protein cleavage site of NDV is a significant deter-minant of NDV virulence and pathogenicity. The strain JL-DH showed a very high ICPI of 1.86, in addition, the strain JL-DH holded 112R-R-Q-K-R-F117 at the cleavage site is in accord-ance with the velogenic strains of NDV [28]. The virulence of NDV is known to be correlative with discrepancy in the amino acid sequence neighboring the post-translational cleavage site of the F0 protein, with discrepancys in the cleavage sites be-ing diametrically related to the virulence of the strain. Most of viruses that are poisonous for chickens have the amino acid sequence 112R/K-R-Q-K/R-R116 at the C-terminus of the F2 protein and F (phenylalanine) at residue 117, the N-terminus of the F1 protein. In comparison, viruses of low virulence have sequences in the identical region of 112G/E-K/R-Q-G/E-R116 and L (leucine) at residue 117 [29,30]. The nucleotide sequence differentiation to the typical genome of each genotype demon-strated that the strain JL-DH showed least identity to the geno-types HLJ363, SG-Liaoning-2009 and Guangxi4-2000. There are the closest genetic relationships between strain JL-DH and HG-Beijing-2009 strains. It has been demonstrated that the virulent isolates of NDV isolated from China during 2006 were classified into genotype, based on the result of the phylogenetic analysis. Newcastle disease virus strain of genotype was a pri-mary genotype being responsible for most Newcastle disease eruptions since the end of last century. The vaccines were used in current market to prevent and control ND of the Mukteswar, B1, F, La Sota or V4 type, which belongs to genotypes and re-spectively, the most wide La Sota belongs to genotype, and is distinct from the now widespread genotype [31,32]. The geno-types and the antigenicity between vaccine strain and epidemic strain have been changed, though the antigenicity of NDV did not change substantially, the vaccine strain could not provide enough preservation to the epidemic strain genotype NDV in the field.

Acknowledgments This work was supported in part by the Na-tional Key Research and Development Program of China (2017YFD0500706), National Natural Science Foundation of China (31570929), Natural Science Foundation of Heilongjiang Province of China (C2017058), Key Project of Research and De-velopment of Shandong Province of China (2016GSF121020), Key Scientific and Technological Planning Project of Harbin (2016AB3BN036) and Project of Graduate Innovative Scientific Research Foundation of Heilongjiang University (YJSCX2017-065HLJU).

Conflict of Interests The authors declare that they have no competing in-terests.

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