BackgroundThe Newcastle disease vir

Background

The Newcastle disease virus (NDV) belongs to genus

Avulavirus within family Paramyxoviridae, order Mono-negavirales [1]. The virus is enveloped with a single-stranded with negative sense RNA genome. The genome is approximately 15 kb in length and follows the “rule of six” which is a pre-requisite for efficient viral replication [2]. The genome (3’ to 5’) encodes for 6 different



proteins, i.e. nucleoprotein (NP), phosphoprotein (P), fusion protein (F), matrix protein (M), hemagglutinin-neuraminidase (HN), and RNA large polymerase (L) protein. The NP, P and M proteins encompass the viral inner surface whereas the L protein constitutes the viral nucleocapsid together with NP and P proteins. The two surface glycoproteins HN and F are responsible for binding to host cell sialic acid receptors and for fusion of the viral envelop to the host cell membrane, respec-tively [3]. As a property of the family, the NDV carries high protein coding capacity, which is further enhanced by the mechanism called “RNA editing”. This

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mechanism results in generation of V and W proteins with one or two guanines (G) insertion, respectively, during the transcription of P gene mRNA [4].

On the basis of conventional in vivo pathogenicity indices for chicken, NDV strains can be divided into pathotypes. The viscerotropic velogenic NDV is highly pathogenic and causes intestinal infection with high mortality, whereas neurotropic velogenic NDV is responsible for symptoms of the respiratory and nervous systems with high mortality. The mesogenic strains are relatively less pathogenic, often with acute respiratory and nervous symptoms but with relatively low mortality. The lentogenic strains of NDV cause mild respiratory tract infections. It is asymptomatic enteric form in which host live longer and it is a privilege to virus for replication and shedding [3]. This difference in patho-genicity is primarily due to differences in the cleavage site within the F protein. This protein is synthesized as a precursor (F0) in non-functional state, which then is cleaved by host proteases into two functionally active polypeptides (F1 and F2). All the mesogenic and velo-

genic strains of NDV carry an amino acid sequence of 112R/K-R-Q-R/K-R-F117 within the F protein whereas lentogenic viruses have 112 G/E-K/R-Q-G/E-R-L117 [5].

On the basis of phylogenetic analysis with the partial hypervariable nucleotide sequences of the F gene, NDV strains have been classified into ten genotypes (I-X). The five genotypes (I, II, III, IV, IX) are considered old (1930-1960) and remaining five genotypes (V, VI, VII, VIII, X) are considered recent (after 1960). However, all have indistinguishable pathogenicity in their hosts. The genotype VI and VII are further divided into seven (VIa-g) and five (VIIa-e) subgenotypes, respectively [6,7]. In parallel, an alternative pattern for NDV classification exists which was initially presented by Aldous et al., [8] while conducting studies on a large number of NDV isolates collected from several countries. According to this criterion, the NDV can be grouped into six distinct genetic lineages (1-6) with several sublineages within them. There are around fifty-five complete genomes of different NDV strains available, which can be divided into three different genome lengths: 15186 nt, 15192 nt and 15198 nt [9- 11].

Newcastle disease (ND) is an OIE notifiable disease and notification of any outbreak is mandatory to the OIE [12]. ND is distributed around the globe and is consistently reported from all the continents. In Pakistan, a sporadic form of the disease exists throughout the year, and only a limited number of outbreaks are officially or unofficially reported annually. Despite the extensive and unrestricted use of imported vaccines, NDV still remains the main poultry disease in both commercial and rural chickens of Pakistan [13]. Incompatibility between field and vaccine strains and generation of novel NDV strains explain this





failure of vaccine. Moreover, the role of rural poultry in the epizootiology of NDV in the country has always remained a mystery. In order to evaluate the degree of genetic diversity of NDV strains circulating in backyard poultry and to estimate the relationships to that of NDV currently circulating in the region, the complete genome of NDV isolated from healthy backyard poultry flocks was characterized genetically, phylogenetically and biologically.

Results

Pathogenicity assessment

Ten out of 12 collected samples from apparently healthy backyard poultry flocks were recovered from specific pathogen free (SPF) embryos and showed heamaggluti-nation (HA) titer. Surprisingly, the isolated virus was found virulent by OIE standard criteria. Chicken/BYP/ Pakistan/2010 exhibited mean death time (MDT) of 49.6 h in embryonated chicken eggs. The intracerebral patho-genicity index (ICPI) value of Chicken/BYP/Pakistan/ 2010 was calculated to be 1.5. These results revealed that Chicken/BYP/Pakistan/2010 was similar to virulent strains of NDV, regarding pathogenicity.

Nucleic acid detection

The positive samples were screened for the presence of Newcastle disease virus by real-time reverse transcrip-tion polymerase chain reaction (rRT-PCR) for the matrix and fusion protein genes. All the ten isolates were found positive with F gene based real-time PCR. However, M gene based real-time PCR failed to detect even a single sample.

Phylogenetic analysis

The phylogenetic relationships of Chicken/BYP/Paki-stan/2010 with other members of NDV were obtained by comparing the nucleotide sequences of the complete coding region of the F gene representing the IX geno-types. The resulting phylogenetic tree is depicted in Fig-ure 1. From the topology of the Bayesian tree, presented in Figure 1, it was apparent that the NDV isolate under study was placed close to genotype VII. However, Chicken/BYP/Pakistan/2010 showed only 89.0% nucleo-tide similarity to that of NA-1 (DQ659677), a represen-tative of genotype VII, when the complete genomes of both isolates were compared. Based on the frequency distribution criteria for genotype classification, Chicken/ BYP/Pakistan/2010 could therefore be considered as separate genotype/subgenotype (Table 1). The existence of deep rooted branching for the isolate and clustering well apart from the rest of sequences within genotype VII provided substantial evidences that support this iso-late as separate genotype/subgenotype. Being a member of genotype VII, Chicken/BYP/Pakistan/2010 clustered


to the isolates from India, Iran and Sweden along with other Pakistani isolates sequenced from 2005-08. In gen-eral, NDV isolates from other Asian countries such as China, Japan and Taiwan constitute genotype VII.


The genotype VII can further be divided into five sub-genotypes (VIIa-e) as presented in Figure 2, and Chicken/BYP/Pakistan/2010 clustered specifically close to subgenotype b (VII-b). However, it showed significant


differences to rest of the subgenotypes and it clustered apart. Hence, tentatively could be considered as a new subgenotype (VII-f). Notably, the Pakistani isolates from previous study also clustered with Chicken/BYP/Paki-stan/2010 within VII-f.

It has been speculated that HN gene can differentiate the genotypes of NDV clearly and may true predict the pathogenicity of the isolates because the length of HN protein varies and cleavage site is not the sole criteria for pathogenicity [11,14]. Therefore, the phylogenetic analysis was conducted using complete coding region of the HN gene. In general, the same topology of the tree was observed as seen with the Bayesian tree of F gene analysis. The Chicken/BYP/Pakistan/2010 clustered

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together in genotype VII as expected, in relation to its HN protein length of 571 aa (Figure 3).

Genomic and non-coding sequence analysis

ND viruses can be divided into groups based on genome lengths, with earlier lineages (I-IV) having a length of 15186 nt, recent lineages (V-VII) a length of 15192 nt due to insertion of 6 nt into the 5’ non-coding region of the NP gene, and the class 1 APMV-1 a length of 15198 nt with the insertion of 12 nt into the coding region of




P gene. The length of Chicken/BYP/Pakistan/2010 was 15192 nt (GenBank accession number JN682210) and is thus follow the “rule of six”, a feature that has been found essential for the effective viral replication. As typi-cal among paramyxoviruses, the genome was organized as NP-P/V/W-M-F-HN-L from 3’ to 5’ end of the gen-ome. The protein coding capacity of the genome was estimated to be 90.4% and the GC content 46%. Among

100 100
99


the full-length sequences of NDV available in the Gen-Bank, Chicken/BYP/Pakistan/2010 showed highest nucleotide sequence similarity with Sterna/Astr/2755/ 2001 (GenBank accession number AY865652). This virus was isolated from a little tern (Sterna albifrons Pallas) in the Volga river delta in Russia.

The genome of APMV-1 starts with a stretch of sequences known as leader at the 3’end, and ends at the trailer sequence at the 5’end. The 3’-leader sequence serves as a promoter from where transcription of mRNA starts and continues through a mechanism com-monly known as “start-stop-restart”. Each gene of the APMV-1 starts with a relatively conserved sequence of gene start (GS) and ends at a sequence of gene end [6].

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The open reading frame (ORF) of each gene overhangs with 3’and 5’ untranslated regions (UTRs) on their respective ends. Between GE of one gene and GS of the next, a conserved sequence exists known as intergenic sequence (IGS). All these features for Chicken/BYP/ Pakistan/2010 are summarized in Table 2. The GS and GE sequences were found to be conserved amon

Hasil (Bahasa Indonesia) 1:

Latar belakangVirus penyakit Newcastle (Power Tools) milik genusAvulavirus dalam keluarga Paramyxoviridae, memesan Mono-negavirales [1]. Virus ditutupi dengan beruntai tunggal dengan pengertian yang negatif genom RNA. Genom adalah kira-kira 15 kb panjang dan mengikuti "aturan enam" yang merupakan pra-syarat untuk replikasi virus efisien [2]. Genom (3' untuk 5') encode 6 berbeda protein, yaitu nucleoprotein (NP), phosphoprotein (P), fusi protein (F), matriks protein (M), hemaglutinin-neuraminidase (HN), dan RNA polimerase besar (L) protein. Protein NP, P dan M mencakup permukaan bagian dalam virus sedangkan L protein merupakan nukleokapsid-sebuah virus dengan protein NP dan P. Glikoprotein permukaan dua HN dan F bertanggung jawab untuk mengikat reseptor sialic acid host sel dan untuk perpaduan viral menyelimuti membran sel inang, masing-tively [3]. Sebagai properti dari keluarga, Power Tools membawa kapasitas pengkodean protein tinggi, yang ditingkatkan dengan mekanisme yang disebut "Pengeditan RNA". Ini Munir et al. virologi jurnal 2012, 9:46 halaman 2 dari 11 http://www.virologyj.com/content/9/1/46 mekanisme hasil dalam generasi V dan W protein dengan satu atau dua guanines (G) penyisipan, masing-masing, selama transkripsi gen P mRNA [4].Berdasarkan konvensional di vivo pathogenicity indeks untuk ayam, Power Tools strain dapat dibagi menjadi pathotypes. Velogenic viscerotropic Power Tools patogenik dan menyebabkan infeksi usus dengan mortalitas tinggi, sedangkan neurotropic velogenic Power Tools bertanggung jawab untuk gejala sistem pernapasan dan gugup dengan mortalitas tinggi. Mesogenic strain yang relatif kurang patogen, sering dengan akut pernapasan dan saraf gejala tetapi dengan kematian relatif rendah. Alunan lentogenic Power Tools menyebabkan infeksi saluran pernapasan ringan. Ini adalah bentuk enterik asimtomatik dalam angkatan yang hidup lagi dan itu suatu kehormatan untuk virus untuk replikasi dan penumpahan [3]. Perbedaan ini di patho-genicity adalah terutama karena perbedaan dalam situs pembelahan dalam F protein. Protein ini disintesis sebagai prekursor (F0) di negara non-fungsional, yang kemudian adalah diurai oleh protease host ke dua akan Proline fungsional aktif (F1 dan F2). Semua mesogenic dan velo-genic strain Power Tools membawa urutan asam amino 112R/K-R-Q-R/K-R-F117 dalam F protein sedangkan lentogenic virus memiliki 112 G/E-K/R-Q-G/E-R-L117 [5].Berdasarkan analisis filogenetik dengan urutan nukleotida parsial hypervariable gen F, Power Tools strain telah diklasifikasikan ke dalam sepuluh genotipe (I-X). Genotipe lima (I, II, III, IV, IX) dianggap tua (1930-1960) dan sisa lima genotipe (V, VI, VII, VIII, X) dianggap sebagai hari (setelah 1960). Namun, semua memiliki pathogenicity tidak dapat dibedakan di host mereka. Genotipe VI dan VII dibagi lagi menjadi tujuh (VIa-g) dan lima (VIIa-e) subgenotypes, masing-masing [6,7]. Secara paralel, pola alternatif untuk Power Tools klasifikasi ada yang dibawakan oleh Aldous et al., [8] sementara melakukan penelitian pada sejumlah besar Power Tools isolat dikumpulkan dari berbagai negara. Menurut kriteria ini, Power Tools dapat dikelompokkan ke dalam enam berbeda genetik keturunan (1-6) dengan beberapa sublineages dalam diri mereka. Ada sekitar lima puluh lima lengkap genom berbeda galur Power Tools yang tersedia, yang dapat dibagi menjadi tiga berbeda genom panjang: 15186 nt, 15192 nt dan 15198 nt [9 - 11].Newcastle disease (ND) adalah penyakit menaati OIE dan pemberitahuan setiap wabah wajib untuk OIE [12]. ND didistribusikan di seluruh dunia dan secara konsisten dilaporkan dari semua benua. Di Pakistan, bentuk sporadis penyakit ada sepanjang tahun, dan hanya sejumlah terbatas wabah resmi atau tidak resmi dilaporkan setiap tahunnya. Meskipun luas dan tidak terbatas penggunaan vaksin impor, Power Tools masih tetap penyakit unggas utama dalam Umum dan BPR ayam Pakistan [13]. Ketidakcocokan antara bidang dan vaksin strain dan generasi baru Power Tools strain menjelaskan hal ini kegagalan vaksin. Selain itu, peran unggas pedesaan di epizootiology Power Tools di negara selalu tetap menjadi misteri. Untuk mengevaluasi tingkat keanekaragaman genetik galur Power Tools yang beredar di backyard unggas dan untuk memperkirakan hubungan dengan Power Tools yang saat ini beredar di wilayah, genom lengkap dari Power Tools yang terisolasi dari sehat backyard unggas kawanan ditandai secara genetik, filogenetis dan biologis.HasilPenilaian pathogenicitySepuluh dari 12 mengumpulkan sampel dari rupanya sehat backyard ternak unggas pulih dari spesifik patogen gratis (SPF) embrio dan menunjukkan titer heamaggluti-negara (HA). Anehnya, terisolasi virus ditemukan virulen oleh OIE kriteria standar. Ayam BYP/Pakistan 2010 dipamerkan berarti kematian waktu (MDT) 49.6 h di telur ayam embryonated. Nilai indeks (ICPI) patho-genicity intraserebral ayam/BYP/Pakistan/2010 dihitung menjadi 1,5. Hasil ini menunjukkan bahwa ayam/BYP/Pakistan/2010 ini mirip dengan strain virulen Power Tools, mengenai pathogenicity.Asam nukleat deteksiSampel positif yang disaring untuk keberadaan virus penyakit Newcastle oleh real-time reverse transcrip-tion PCR (rRT-PCR) untuk gen protein matriks dan fusion. Semua sepuluh isolat tersebut ditemukan positif dengan F gen berdasarkan real-time PCR. Namun, gen M berdasarkan real-time PCR gagal mendeteksi bahkan satu contoh.Analisis filogenetikHubungan filogenetik ayam/BYP/Paki-stan/2010 dengan anggota lain dari Power Tools diperoleh dengan membandingkan urutan nukleotida daerah coding lengkap gen F mewakili IX geno-jenis. Pohon filogenetik dihasilkan digambarkan dalam ara-ure 1. Dari topologi Bayesian pohon, disajikan dalam gambar 1, sudah jelas bahwa isolat Power Tools di bawah study ditempatkan dekat dengan genotipe VII. Namun, ayam/BYP/Pakistan/2010 menunjukkan kesamaan 89.0% nucleo pasang hanya bahwa NA-1 (DQ659677), perwakilan tative dari genotipe VII, ketika genom lengkap dari kedua isolat dibandingkan. Berdasarkan kriteria distribusi frekuensi klasifikasi genotipe, ayam / BYP/Pakistan/2010 karena itu dianggap sebagai terpisah genotipe/subgenotype (Tabel 1). Keberadaan mendalam berakar bercabang untuk isolat dan pengelompokan juga terpisah dari sisa urutan dalam genotipe VII disediakan substansial bukti-bukti yang mendukung iso akhir sebagai genotipe/subgenotype terpisah. Menjadi anggota genotipe VII, berkerumun ayam/BYP/Pakistan/2010 untuk isolat tersebut dari India, Iran, dan Swedia dengan lain isolat Pakistan yang diurutkan dari 2005-08. Dalam gen-memeriksa, Power Tools isolat dari negara-negara Asia seperti Cina, Jepang dan Taiwan merupakan genotipe VII. Genotipe VII dapat lebih lanjut dibagi menjadi lima sub genotipe (VIIa-e) yang disajikan dalam gambar 2, dan ayam/BYP/Pakistan/2010 berkerumun khususnya dekat dengan subgenotype b (VII-b). Namun, hal ini menunjukkan signifikan perbedaan untuk beristirahat subgenotypes dan tersusun terpisah. Oleh karena itu, tentatif dapat dianggap sebagai subgenotype baru (VII-f). Terutama, isolat Pakistan sebelumnya studi juga berkerumun dengan ayam/BYP/Paki-stan/2010 dalam VII-f.Telah speculated bahwa gen HN dapat membedakan genotipe Power Tools jelas dan benar dapat memprediksi pathogenicity isolat tersebut karena panjang HN protein bervariasi dan situs pembelahan bukanlah satu-satunya kriteria untuk pathogenicity [11,14]. Oleh karena itu, analisis filogenetik dilakukan menggunakan lengkap wilayah pengkodean gen HN. Secara umum, topologi yang sama pohon diamati seperti yang terlihat dengan pohon Bayesian F gen analisis. Berkerumun ayam/BYP/Pakistan/2010 Halaman 4 dari 11bersama-sama dalam genotipe VII seperti yang diharapkan, dalam kaitannya dengan panjangnya protein HN 571 aa (gambar 3).Analisis urutan genom dan bebas-codingVirus ND dapat dibagi menjadi kelompok-kelompok yang didasarkan pada genom panjang, dengan keturunan awal (I-IV) memiliki panjang 15186 nt, hari keturunan (V-VII) panjang 15192 nt karena penyisipan 6 nt ke 5' bebas-coding wilayah NP gen, dan kelas 1 APMV-1 panjang 15198 PB dengan penyisipan 12 nt ke pengkodean wilayah Gen P. Panjang ayam/BYP/Pakistan/2010 adalah 15192 nt (GenBank aksesi nomor JN682210) dan dengan demikian mengikuti "aturan enam", sebuah fitur yang telah ditemukan penting untuk replikasi virus yang efektif. Sebagai typi-cal antara paramyxoviruses, genom diselenggarakan sebagai NP-P/V/W-M-F-HN-L dari 3' 5' akhir gen-ome. Kapasitas pengkodean protein genom diperkirakan 90,4% dan konten GC 46%. Antara 100 100 99 urutan-urutan full-length Power Tools tersedia dalam Gen-Bank, ayam/BYP/Pakistan/2010 menunjukkan kemiripan urutan nukleotida tertinggi dengan 2755/Sterna/Astr/2001 (GenBank aksesi nomor AY865652). Virus ini adalah terisolasi dari Dara-laut kecil (Sterna albifrons Pallas) di delta Sungai Volga di Rusia.Genom dari APMV-1 dimulai dengan bentangan urutan yang dikenal sebagai pemimpin di ujung 3', dan berakhir pada urutan trailer di ujung 5'. Urutan 3'-pemimpin berfungsi sebagai promotor dari mana mRNA transkripsi mulai dan terus melalui mekanisme com-monly yang dikenal sebagai "start-stop-restart". Setiap gen APMV-1 dimulai dengan urutan relatif dilestarikan gen mulai (GS) dan berakhir pada urutan gen akhir [6]. Munir et al. virologi jurnal 2012, 9:46 Halaman 5 dari 11http://www.virologyj.com/content/9/1/46Frame terbuka membaca (ORF) gen setiap serta overhang dengan 3' 5' diterjemahkan daerah (UTRs) di masing-masing ujung. Antara GE satu gen dan GS berikutnya, urutan dilestarikan ada dikenal sebagai intergenic urutan (IGS). Semua fitur ini untuk ayam BYP/Pakistan 2010 diringkas dalam tabel 2. Urutan GS dan GE ditemukan harus dilestarikan amon