The open reading frame (ORF) of eac

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 among the genes. An identical GS (ACG3TAGA2) was observed for the first five genes (NP, P, M, F, and HN), whereas one nucleotide difference (ACG3TAG2A) was seen in the GS of the last gene (L). The GE sequence in the NP, M, HN and L genes (T2AGA6) was different from that in the P and F genes (T2A2GA6). This feature is common among NDV strains of genotype VII [11].

The 3’-terminus sequence (1-19 nt) and 5’-terminus sequence (15173-15192 nt) of Chicken/BYP/Pakistan/ 2010 was uncertain because the primers were designed based on the consensus generated by the alignment of the 52 complete genomes. However, the comparison of these sequences revealed that both termini are con-served especially the first 12 nt and last 8 nt in the 3’and 5’ends of the genome, respectively.

Features of the coding region

NP gene analysis

The NP gene of Chicken/BYP/Pakistan/2010 showed a highest nucleotide and amino acid similarity to that of NA-1 (DQ659677) and IT-227/82 (AJ880277), respec-tively (Table 3) when compared to representatives of each genotype. The other features of all the genes such as gene length, ORF length, GS, GE and predicted mole-cular weight are summarized in Table 2. All APMV-1 carries a stretch of 15 amino-acids (N’-FX4YX3FS-FAMG-C’, where × is any amino acid and F is any aro-matic amino acid), in the middle of NP ORF, which is responsible for the N-N self assembly in the process of RNA binding and this sequence in Chicken/BYP/Paki-stan/2010 was 322FAPAEYAQLYSFAMG336.



P gene analysis

The P protein is the only multi-coding protein of NDV, identified so far. The P gene undergo RNA editing due to stuttering of the polymerase complex over the tem-plate and leads to insertion of non-templated nucleotide

(s) (guanine, G) in the editing site. The editing site (AAAAAGGG) in Chicken/BYP/Pakistan/2010 was found at position 394-401. The P gene transcribe into P protein (unedited), V protein (+1 frame-shift) and W protein (+2 frame-shift). These three proteins share N terminus and have unique C-terminus [4]. The P gene was the most variable gene among the six NDV genes when compared with representatives of each genotype. Therefore, it is believed that the P gene is an evolution-ary strategy of the virus that increases the coding capa-city of the genome [16]. The lengths of the V proteins of almost all NDV strains including Chicken/BYP/Paki-stan/2010 are identical (239 aa). Comparison of the V protein from representative NDVs from each lineage revealed that the C-terminus of the V protein is highly conserved and this starts from the most variable region of editing site [11,17]. The seven cysteine residues, which have been identified in the C-terminus are responsible for the co-ordination of two zinc atoms to form a unique finger fold. These residues were located at positions 196, 200, 212, 214, 217, 221 and 224 in Chicken/BYP/Pakistan/2010. However, the length of W protein is highly variable (147 aa to 227 aa). Our sequencing found a length of 227 aa for the W protein of Chicken/BYP/Pakistan/2010. It has been shown that the length of W protein is not associated with any viru-lence of NDV [18].

M gene analysis

The M protein of NDV is primarily involved in the assembly and intracellular transport of NDV compo-nents. Sequencing in our study indicated that the length of the M protein of Chicken/BYP/Pakistan/2010 was 364 aa which is identical to all other NDV strains except

Herts/33 with a length of 380 aa. The M protein of indicated that the NDV strain Beaudette C if mutated at
NDV is localized in the nucleus and this localization is position 526 (Y526Q) reduced the neuraminidase recep-
mediated through nuclear localization signals (NLS). tor binding and fusion activities of NDV and thus lead
The NLS 246DKKGKKVTFDKIEEKIRR263 was identified to attenuation of viral virulence in eggs and young birds.
for Chicken/BYP/Pakistan/2010 as for other strains of It has been observed that there are seven antigenic
NDV. sites within HN protein that are involved in the forma-
HN gene analysis tion of a continuum in the three dimensional conforma-
The HN protein of NDV is a multifunctional protein tion of HN molecules as demonstrated in Table 5. A

that plays a crucial role in virus infectivity. The primary total of five substitutions, I514V, D569V, N263K, E347D
role of the HN protein is to bind with sialic acid con- and R353Q have been observed in region 2, 3 and 14 of
taining receptors, which play key role in the initiation of the HN protein of Chicken/BYP/Pakistan/2010. There
viral infection in the host. Special emphasis was given to have been five conserved potential glycosylation sites
HN protein due to the isolation of Chicken/BYP/Paki- identified at position 119 (NNSG), 341 (NNTC), 433
stan/2010 from healthy backyard poultry flocks. In order (NKTA), 481 (NHTL) and 538 (NKVY) whereas one site
to understand the contribution of HN protein in recep- at position 508 was absent in Chicken/BYP/Pakistan/
tor specificity, the analysis of deduced amino acid 2010 which is considered non-conserved among para-
sequence of HN protein was performed. Out of 14 resi- myxoviruses [20].

dues (174, 175, 198, 236, 258, 299, 317, 401, 416, 498, There have been three lengths reported for the HN
516, 526 and 547) identified essential for the receptor protein of NDV, which depends upon genotype. The
recognition, Chicken/BYP/Pakistan/2010 showed a sub- lengths of HN protein in genotype I is 616 aa, in geno-
stitution at 526 (Y526Q) (Table 4 and Figure 4). Inter-
type II is 577 aa, whereas the HN length in genotypes
estingly, a study conducted by Khattar et al., [19]
IV, V and VII are 571 aa, irrespective of their


















pathogenicity. As expected, being a genotype VII, Chicken/BYP/Pakistan/2010 had a HN length of 571 aa.

F gene analysis

The F protein is initially synthesized as an inactive pre-cursor (F0), which is cleaved by host-cell proteases into F1 and F2, which constitute biologically active proteins, still connected through disulfide-linked chains. The pri-mary function of the F protein is to initiate the fusion of viral surface to that of host cell membrane [3]. As a

rule of thumb, a consensus amino acid sequence of 112R/K-R-Q-R/K-R-F117 is present in velogenic strains of NDV and 112 G/E-K/R-Q-G/E-R-L117 is present in len-
togenic viruses. Despite the fact that Chicken/BYP/Paki-

stan/2010 was isolated from healthy backyard poultry flocks, it contains 112R-R-Q-K-R-F117, which corre-

sponds to the cleavage site of velogenic viruses and was in accordance with its MDT and ICPI.

L gene analysis

The L protein is the largest protein among the six pro-teins of NDV and function as a RNA-dependent-RNA





polymerase. A motif (GDNQ) is considered responsible for the polymerase activity and is highly conserved within L proteins of non-segmented, negative-sense RNA viruses. In Chicken/BYP/Pakistan/2010, a corre-sponding sequence was found (GDNQ) at position 750-753 aa. In comparison with representatives of each gen-otype, the L protein of Chicken/BYP/Pakistan/2010 was found to be the most conserved protein among the six proteins of NDV.

Discussion

This study presents the first characterization of an NDV isolated from rural poultry in Pakistan, and can serve as a basis for future vaccine design. The study highlights the importance of rural poultry in the epizootiology of the disease in the country. According to the criteria set by the OIE, the virulence of NDV is associated with ICPI and the cleavage site in the F protein [12,21]. ICPI of 0.7 or greater in day-old chicken or presence of three basic amino acids (R or K) at the F protein cleavage sits between residues 113 and 116 indicate the virulent form of NDV. The isolate in current study surprisingly exhib-ited MDT of 49.6 h in embryonated chicken eggs and ICPI value of 1.5. Moreover, the Chicken/BYP/Pakistan/ 2010 possessed 112R-R-Q-K-R-F117 at the cleavage site, the same as of the velogenic strains of NDV [5]. All these facts indicate that the rural poultry can harbour virulent strains of NDV without showing clinical signs, and that it consequently may act as silent carriers and constitute a potential threat to the commercial poultry. On the other hand, this finding also indicates that local breeds are more resistant and can sustain the virulent form of the disease. However, the presence of other sec-ondary infections (avian influenza along with other viral, bacterial or parasitic infections) and immuno-compro-mising husbandry factors may provoke the disease, which warrant further studies in the rural poultry chick-ens. Beside these facts, sample collection during incuba-tion period of the disease, can’t be ruled out in this


study. Currently, there is great wealth of reports discuss-ing the error prone genome transcription during replica-tion of RNA viruses such as NDV. This process helps viruses escape host immune defences, alter pathogenicity and host range, and evade diagnostic tests. To properly understand these mechanisms, it is of paramount impor-tance to fully characterize the viruses in order to study within-host dynamics and genetic variation, relate dynamics and variation to transmission, and to recon-struct transmission trees at high resolution.

It has been demonstrated that virulence of NDV is a multigenic trait as that of influenza viruses which is mainly contributed by HN, V and L proteins of NDV [22- 24]. The essential role of L protein in pathogenicity can be shown by e.g. the Beaudette C (BC) strain of NDV, which if it carries the L protein of LaSota increases its

Hasil (Bahasa Indonesia) 1:

Frame 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 dilestarikan antara Gen-gen. GS identik (ACG3TAGA2) diamati untuk gen pertama lima (NP, P, M, F, dan HN), sedangkan nukleotida satu perbedaan (ACG3TAG2A) terlihat di GS gen terakhir (L). Urutan GE dalam gen NP, M, HN dan L (T2AGA6) adalah berbeda dari yang dalam gen P dan F (T2A2GA6). Fitur ini umum di antara strain Power Tools genotipe VII [11].3'-terminus urutan (1-19 PB) dan 5'-terminus urutan (15173-15192 PB) ayam/BYP/Pakistan/2010 adalah pasti karena yang primers dirancang berdasarkan konsensus yang dihasilkan oleh penyelarasan genom lengkap 52. Namun, perbandingan urutan ini mengungkapkan bahwa kedua termini con-disajikan terutama pertama 12 nt dan terakhir 8 nt di 3' dan 5' berakhir genom, masing-masing.Fitur daerah codingAnalisis gen NPGen NP ayam/BYP/Pakistan/2010 menunjukkan nukleotida tertinggi dan asam amino persamaan yang NA-1 (DQ659677) dan IT-227/82 (AJ880277), masing-tively (Tabel 3) bila dibandingkan dengan wakil-wakil dari setiap genotipe. Fitur lain dari semua gen seperti gen panjang, panjang ORF, GS, GE dan berat mol-cular diperkirakan diringkas dalam tabel 2. APMV-1 semua membawa bentangan 15-asam amino (N' - FX4YX3FS - FAMG-C', mana × adalah asam amino dan F adalah asam amino aro-matic), di tengah-tengah NP ORF, bertanggung jawab untuk N-N diri perakitan dalam proses pengikatan RNA dan urutan ini di ayam/BYP/Paki-stan/2010 adalah 322FAPAEYAQLYSFAMG336. Analisis gen PP protein adalah protein hanya multi-coding Power Tools, diidentifikasi sejauh ini. Gen P menjalani RNA mengedit karena gagap dari polimerase kompleks tem-piring dan mengarah ke penyisipan bebas-kerangka nukleotida(s) (guanina, G) di situs editing. Mengedit situs (AAAAAGGG) dalam ayam/BYP/Pakistan/2010 ditemukan pada posisi 394-401. Gen P menuliskan ke protein P (diedit), protein V (+ 1 frame-shift) dan W protein (+ 2 bingkai-shift). Protein ini tiga berbagi N terminus dan memiliki unik C-terminus [4]. Gen P adalah gen paling variabel antara enam Gen-gen Power Tools bila dibandingkan dengan wakil-wakil genotipe masing-masing. Oleh karena itu, hal ini diyakini bahwa gen P adalah strategi evolusi-ary virus yang meningkatkan pengkodean capa-kota genom [16]. Panjang protein V hampir semua galur Power Tools termasuk ayam/BYP/Paki-stan/2010 identik (239 aa). Perbandingan protein V dari perwakilan NDVs dari silsilah setiap mengungkapkan bahwa ujung C V protein sangat kekal dan ini mulai dari wilayah paling variabel mengedit situs [11,17]. Residu Sistein tujuh, yang telah diidentifikasi di ujung C bertanggung jawab koordinasi dua atom seng untuk membentuk sebuah lipatan jari yang unik. Residu ini berada pada posisi 196, 200, 212, 214, 217, 221 dan 224 di ayam/BYP/Pakistan/2010. Namun, panjang protein W sangat bervariasi (147 aa untuk 227 aa). Sekuensing kami menemukan panjang 227 aa untuk protein W ayam/BYP/Pakistan/2010. Telah terbukti bahwa panjang W protein ini tidak terkait dengan setiap viru-mukan penyebab terjadinya kekerasan dari Power Tools [18].Analisis gen MProtein M Power Tools yang terutama terlibat dalam perakitan dan transportasi intraseluler Power Tools compo-nents. Sekuensing dalam penelitian kami menunjukkan bahwa panjang protein M ayam/BYP/Pakistan/2010 364 strain aa yang identik dengan Power-Tools lain kecuali Herts/33 dengan panjang 380 aa. M protein menunjukkan bahwa Power Tools ketegangan Beaudette C jika bermutasi diPower Tools dilokalisasi dalam inti dan lokalisasi ini adalah posisi 526 (Y526Q) berkurang neuraminidase recep-dimediasi melalui sinyal lokalisasi nuklir (NLS). Tor kegiatan mengikat dan fusion Power Tools dan dengan demikian menyebabkanNLS 246DKKGKKVTFDKIEEKIRR263 diidentifikasi untuk redaman virus virulensi telur dan burung muda.untuk ayam/BYP/Pakistan/2010 untuk lain strain telah diamati bahwa terdapat tujuh antigenPOWER TOOLS. situs dalam HN protein yang terlibat dalam forma-Analisis gen HN tion dari sebuah kontinum dalam tiga dimensi conforma -Protein HN Power Tools adalah sebuah tion multifungsi protein HN molekul seperti yang ditunjukkan dalam tabel 5. Ayang memainkan peran penting dalam penularan virus. Total utama substitusi lima, I514V, D569V, N263K, E347Dperan HN protein untuk mengikat dengan asam sialic con - dan R353Q telah diamati di wilayah 2, 3 dan 14taining reseptor, yang memainkan peran kunci dalam inisiasi protein HN ayam/BYP/Pakistan/2010. Adainfeksi virus di dalam angkatan surgawi. Penekanan khusus diberikan kepada telah lima dilestarikan potensi glikosilasi situsHN protein karena isolasi ayam/BYP/Paki - diidentifikasi pada posisi 119 (NNSG), 341 (NNTC), 433Stan 2010 dari sehat backyard unggas kawanan. Dalam rangka (NKTA), 481 (NHTL) dan 538 (NKVY) sedangkan satu situsuntuk memahami kontribusi HN protein dalam recep-pada posisi 508 tidak hadir di Pakistan/ayam/BYP /Tor kekhususan, analisis disimpulkan asam amino 2010 yang dianggap bebas dilestarikan antara para-urutan protein HN dilakukan. Keluar dari 14 resi-myxoviruses [20]. iuran (174, 175, 198, 236, 258, 299, 317, 401, 416, 498, ada tiga panjang yang dilaporkan untuk HN516, 526 dan 547) mengidentifikasi penting untuk reseptor protein Power Tools, yang tergantung pada genotipe. Thepengakuan, ayam/BYP/Pakistan/2010 menunjukkan sub-panjang HN protein dalam genotipe saya adalah 616 aa, geno-gagasan Konstitusi di 526 (Y526Q) (Tabel 4 dan gambar 4). Inter-tipe II adalah 577 aa, sedangkan HN panjang dalam genotipeestingly, sebuah studi yang dilakukan oleh Khattar et al., [19]IV, V, dan VII yang 571 aa, penyesatan dari merekapathogenicity. Seperti yang diharapkan, menjadi genotipe VII, ayam/BYP/Pakistan/2010 memiliki panjang HN 571 aa.Analisis gen FF protein awalnya disintesis sebagai aktif pra-kursor (F0), yang diurai oleh protease sel inang ke F1 dan F2, yang merupakan protein biologis aktif, masih terhubung melalui rantai disulfida-terkait. Fungsi F protein pri-Maria adalah untuk memulai fusi dari permukaan virus dari membran sel inang [3]. Sebagaiaturan, konsensus urutan asam amino 112R/K-R-Q-R/K-R-F117 adalah hadir dalam velogenic strain Power Tools dan 112 G/E-K/R-Q-G/E-R-L117 hadir dalam len-virus togenic. Terlepas dari kenyataan bahwa ayam/BYP/Paki-Stan 2010 diasingkan dari sehat backyard unggas kawanan, mengandung 112R-R-Q-K-R-F117, edisi yang-sponds ke situs pembelahan velogenic virus dan sesuai dengan MDT dan ICPI.Analisis gen LL protein adalah protein yang terbesar di antara enam pro-teins Power Tools dan fungsi sebagai RNA-dependent-RNA polimerase. Motif (GDNQ) dianggap bertanggung jawab untuk aktivitas polimerase dan sangat kekal dalam L protein virus RNA bebas tersegmentasi, negatif-rasa. Ayam/BYP/Pakistan/2010, urutan edisi-sponding adalah menemukan (GDNQ) di posisi 750-753 aa. Dibandingkan dengan wakil-wakil dari setiap gen-otype, protein L ayam/BYP/Pakistan/2010 ditemukan untuk menjadi paling dilestarikan protein antara enam protein dari Power Tools.DiskusiStudi ini menyajikan karakterisasi pertama Power Tools yang terisolasi dari pedesaan unggas di Pakistan, dan dapat berfungsi sebagai dasar untuk masa depan vaksin desain. Studi ini menyoroti pentingnya unggas pedesaan di epizootiology penyakit di negara. Sesuai dengan kriteria yang ditetapkan oleh OIE, virulensi Power Tools ini dikaitkan dengan ICPI dan situs pembelahan protein F [12,21]. ICPI 0.7 atau lebih hari-tua ayam atau kehadiran tiga dasar asam amino (R atau K) di F protein pembelahan duduk antara residu 113 dan 116 menunjukkan bentuk virulen Power Tools. Isolat saat ini studi mengejutkan exhib-ited MDT 49.6 h di telur ayam embryonated dan nilai ICPI 1.5. Selain itu, ayam/BYP/Pakistan/2010 dimiliki 112R-R-Q-K-R-F117 di situs pembelahan, sama seperti alunan velogenic Power Tools [5]. Semua fakta-fakta ini menunjukkan bahwa unggas pedesaan dapat pelabuhan strain virulen Power Tools tanpa menunjukkan tanda-tanda klinis, dan bahwa itu akibatnya dapat bertindak sebagai pembawa diam dan merupakan potensi ancaman terhadap unggas. Di sisi lain, temuan ini juga menunjukkan bahwa bibit lokal lebih tahan dan dapat mempertahankan bentuk virulen penyakit. Namun, adanya infeksi sec-ondary (flu bersama dengan infeksi virus, bakteri atau parasit lainnya) dan peternakan immuno-compro-mising faktor lain mungkin memprovokasi penyakit, yang menjamin lebih lanjut studi di unggas pedesaan chick-ens. Di samping fakta-fakta ini, pengambilan sampel selama periode incuba-tion penyakit, tidak dapat dikesampingkan dalam hal ini studi. Saat ini, ada kekayaan besar laporan membahas-ing transkripsi genom rawan kesalahan selama replika-tion virus RNA seperti Power Tools. Proses ini membantu virus melarikan diri pertahanan kekebalan inang, mengubah pathogenicity dan inang, dan menghindari tes diagnostik. Untuk memahami mekanisme ini benar, itu adalah impor-bantuan penting untuk sepenuhnya menandai virus untuk mempelajari dinamika dalam host dan variasi genetik, berhubungan dinamika dan variasi transmisi, dan pohon-pohon transmisi recon-struct pada resolusi tinggi.Telah ditunjukkan bahwa virulensi Power Tools adalah sifat multigenic seperti virus influenza yang terutama disumbangkan oleh HN, V dan L protein dari Power Tools [22-24]. Peran penting L protein dalam pathogenicity dapat ditampilkan oleh misalnya ketegangan Beaudette C (SM) Power Tools, yang jika ia membawa L protein LaSota meningkatkan nya