DiscussionThis study presents the f

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 virulence. Recombinant viruses deficient in V protein have a tendency to be attenuated and show impaired growth in cell culture, an indication of V pro-tein involvement in the virulence of the virus by down regulation of the host immune response [24]. Applying reverse genetics, it has been concluded that BC strains show decreased virulence when HN protein was replaced with LaSota, and that the virulence of LaSota strain was increased when HN protein was replace with HN protein of BC. Moreover, the role of 5’UTR of the HN gene has been shown to be essential for the patho-genicity of the virus. The HN protein of Chicken/BYP/ Pakistan/2010 showed an Y526Q substitution at the receptor-binding site. This site has been demonstrated essential for the neuraminidase receptor binding, and fusion activities of the NDV [19]. This substitution leads to reduction in the pathogenicity of NDV both in vitro and in vivo. This fact might explain the reduced or atte-nuated pathogenicity of the Chicken/BYP/Pakistan/2010 in rural poultry birds. However, it is to mention that the isolate regained the pathogenicity when chicken embryos were infected and this may demand further studies to look for this mechanism at molecular level.

The isolate under study was classified in genotype VII, which is the pre-dominant genotype responsible for ND outbreaks in the Asian countries, including Pakistan. However, a previous study conducted on NDV isolates from Karachi showed that at least two different geno-types (VI and VII) are present in the country [13]. All these isolates were clustered with isolates from China and India suggesting they were derived from common sources and spread between the countries and the role of wild bird population cannot be ignored. It has been reported before that genetically distinct genotypes may co-circulate in a region and cause disease [25]. Genotype VII is predominantly reported from Asian countries such as Korea, Taiwan and China since 1980. The F gene based analysis suggested that Chicken/BYP/





Pakistan/2010 is closest to the Chinese NDV isolates among bordering countries. It can be mentioned that the H5N1 highly pathogenic influenza viruses in Paki-stan are also genetically related to isolates from China [15,26]. There could be several possibilities, based on ground realities, for this fact. The movement of con-taminated material and illegal infected material across the border may be the main factor in this transmission. The wild birds may also play role in dissemination of these viruses [27]. Therefore, the extensive surveillance of the wild birds for the NDV is a fundamental require-ment for understanding the epidemiology of this virus as it has usually been practiced for avian influenza viruses.

Several form of vaccines carrying different strains of NDV (F, LaSota and Mukteswar) have been used in Pakistan [28]. The complete genome sequence compari-son to the representative genome of each genotype revealed that Chicken/BYP/Pakistan/2010 showed least identity to the LaSota (AY845400) and Mukteswar (EF201805) strain with percentage identity of 84.0 and 85.0, respectively. Being grouped in lineage VII, NA-1 (DQ659677) showed highest identity to Chicken/BYP/ Pakistan/2010. Among all the complete genomes avail-able in the GenBank, Chicken/BYP/Pakistan/2010 was highly identical to that of Sterna/Astr/2755/2001 (Gen-Bank accession number AY865652). This isolate is iden-tified from little Tern (Sterna albifrons Pallas) in the Volga river delta in Russia. Considering the transbound-ary nature of the disease, Chicken/BYP/Pakistan/2010 showed 85.2% and 84.3% sequence identity to two Indian isolates, NDV-4 (HM357251) and NDV2K17 (HQ902590), respectively. To our knowledge, there is no complete genome sequence of NDV available from other countries in the region such as Iran, Afghanistan, Ban-gladesh and Nepal.

In spite of extensive potential of sequence data evalua-tion in epizootiological investigations, the information on the geographical distribution of epizootic NDV geno-types is extremely limited and mainly insufficient for epidemiological investigations, especially in the South-East Asian countries. Moreover, to establish epidemiolo-gical link and to understand introduction of ND to a new location (as of European NDV strains-genotype VII), it is fundamental requirement to characterize the viruses in the regions where this data is lacking. It is even more important in country like Pakistan being major exporter of wild birds to the EU, sending tens of thousands of parrots and other birds destined for Eur-opean pet markets each year, particularly to Italy, Spain, Portugal, and Greece [29]. Recently, it has been observed that the parrots, lovebirds and finches imported from Pakistan to Italy were carrying exotic Newcastle disease [29]. Such facts impose national






collections of NDV strains, which would prove useful in future epidemiological investigations.

To conclude, we have characterized a full-length gen-ome of a velogenic NDV from healthy backyard poultry flock, which belongs to the genotype VII along with other Asian isolates. We have provided evidence for the existence of novel genetic group of NDV in Pakistan and that substantial changes in the probe site of M-gene based real-time PCR. This isolate will be valuable in analyzing the genetic nature of APMV-1 not only in Pakistan, but also in other neighboring countries. Find-ings in this study advance the currently available full genome data on APMV-1. Furthermore, we have estab-lished a domestic reference virus for future studies which would lead to development and selection of appropriate strain of NDV for vaccine.

Methods

Virus collection and isolation

In an attempt to screen for pathogens, cloacal swabs, tracheal swabs and blood samples were collected from apparently healthy poultry flocks kept at homes of farm-assistants working in nearby commercial poultry farms. Samples from individual birds were inoculated in five 10-day-old specific pathogen free chicken embryos via allantoic cavity. Three days after inoculation, the allan-toic fluid was harvested and clarified by centrifugation at 4000 × g for 30 min at 4°C. The supernatant was col-lected and used to run for standard hemagglutination inhibition test using specific antisera to the reference strains of NDV (avian paramyxovirus type I). Samples showing high HA titer were divided into working stocks and stored at -20°C. These allantoic fluids were used in pathogenicity assessment and for sequence analysis. For genome detection and characterization, allantoic fluid from each flock was stored on QIAcard FTA Indicator Four Spots (Qiagen, Hilden, Germany), which preserve nucleic acids and inactivate the virus. The samples were shipped at ambient temperature from Pakistan to the Swedish University of Agricultural Sciences, Uppsala, Sweden, for processing.

Intracerebral pathogenicity index (ICPI)

ICPI was performed in ten 1-day old chicks by inocula-tion of 50 μl of allantoic fluid with hemagglutination (HA) titer of more than 24 and diluted 10 fold in PBS without antibiotics, as recommended [21]. The birds were kept under observation for one week and exam-ined after each 24 h. The birds scored an ICPI of 0.7 were declared as lentogenic strain of NDV and the birds with ICPI of more than 1.5 were considered velogenic. The NDV strains with intermediate ICPI values were designated as mesogenic.





Mean death time (MDT) calculation

For MDT, allantoic fluid carrying viru

Hasil (Bahasa Indonesia) 1:

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 ditunjukkan oleh misalnya ketegangan Beaudette C (SM) Power Tools, yang jika ia membawa L protein LaSota meningkatkan virulensi nya. Rekombinan virus kekurangan protein V memiliki kecenderungan untuk menjadi dilemahkan dan menunjukkan gangguan pertumbuhan pada kultur sel, indikasi keterlibatan pro-tein V dalam virulensi virus oleh bawah peraturan respon imun inang [24]. Menerapkan genetika terbalik, itu telah telah menyimpulkan bahwa BC strain menunjukkan penurunan virulensi ketika HN protein diganti dengan LaSota, dan bahwa virulensi LaSota ketegangan meningkat ketika HN protein ganti dengan protein HN BC. Selain itu, peran 5' UTR HN gen telah terbukti menjadi penting untuk patho-genicity dari virus. Protein HN ayam BYP/Pakistan 2010 menunjukkan substitusi Y526Q di situs mengikat reseptor. Situs ini telah dibuktikan penting untuk mengikat reseptor neuraminidase, dan kegiatan fusi Power Tools [19]. Penggantian ini mengarah ke pengurangan dalam pathogenicity Power Tools secara in vitro dan in vivo. Kenyataan ini mungkin menjelaskan dikurangi atau atte-nuated pathogenicity ayam/BYP/Pakistan/2010 di pedesaan unggas burung. Namun, adalah untuk menyebutkan bahwa isolat kembali pathogenicity ketika ayam embrio yang terinfeksi dan ini mungkin menuntut lebih lanjut studi untuk mencari mekanisme ini pada tingkat molekuler.Isolat diteliti diklasifikasikan dalam genotipe VII, yang merupakan pra-dominan genotipe bertanggung jawab untuk ND wabah di negara-negara Asia, yang termasuk Pakistan. Namun, sebuah penelitian sebelumnya yang dilakukan pada Power Tools isolat dari Karachi menunjukkan bahwa setidaknya dua geno-jenis (VI dan VII) hadir di negara [13]. Ternyata semua isolat tersebut berkumpul dengan isolat dari Cina dan India menyarankan mereka berasal dari sumber Umum dan tersebar antara negara-negara dan peran populasi burung liar tidak dapat diabaikan. Telah dilaporkan sebelumnya bahwa genotipe berbeda secara genetik mungkin Co beredar di wilayah dan menyebabkan penyakit [25]. Genotipe VII didominasi dilaporkan dari negara-negara Asia seperti Korea, Taiwan dan Cina sejak tahun 1980. Gen F berbasis analisis menyarankan bahwa ayam/BYP / Pakistan 2010 yang terdekat Power Tools Cina isolat tersebut antara negara yang berbatasan. Itu bisa disebutkan bahwa virus patogenik flu H5N1 di Paki stan juga genetik berkaitan isolat dari Cina [15,26]. Mungkin ada beberapa kemungkinan, berdasarkan tanah realitas, untuk fakta ini. Gerakan con-taminated bahan dan materi terinfeksi ilegal melintasi perbatasan mungkin menjadi faktor utama dalam transmisi ini. Burung liar juga mungkin memainkan peran dalam penyebaran virus ini [27]. Oleh karena itu, pengawasan ekstensif burung liar untuk Power Tools adalah fundamental memerlukan-ment untuk memahami epidemiologi virus ini seperti itu biasanya telah dipraktekkan selama virus flu burung.Beberapa bentuk vaksin yang membawa strain yang berbeda dari Power Tools (F, LaSota dan Mukteswar) telah digunakan di Pakistan [28]. Lengkap genom urutan compari-anak genom perwakilan dari setiap genotipe mengungkapkan bahwa ayam/BYP/Pakistan/2010 menunjukkan identitas setidaknya LaSota (AY845400) dan Mukteswar (EF201805) ketegangan dengan persentase identitas 84.0 dan 85.0, masing-masing. Yang dikelompokkan dalam keturunan VII, NA-1 (DQ659677) menunjukkan identitas tertinggi untuk ayam BYP/Pakistan 2010. Di antara semua lengkap genom berhasil-dapat di GenBank, ayam/BYP/Pakistan/2010 adalah sangat identik dengan yang Sterna/Astr/2755/2001 (Gen-Bank aksesi nomor AY865652). Isolat ini adalah iden-tified dari Dara-laut kecil (Sterna albifrons Pallas) di delta Sungai Volga di Rusia. Mengingat sifat transbound-ary penyakit, ayam/BYP/Pakistan/2010 menunjukkan 85,2% dan 84,3% urutan identitas untuk dua isolat India, Power Tools-4 (HM357251) dan NDV2K17 (HQ902590), masing-masing. Untuk pengetahuan kita, ada tidak ada urutan genom lengkap Power Tools tersedia dari negara lain di wilayah seperti Iran, Afghanistan, Ban-gladesh dan Nepal.Meskipun luas potensi urutan data evalua-tion dalam penyelidikan epizootiological, informasi tentang distribusi geografis epizootic Power Tools geno-jenis sangat terbatas dan terutama mencukupi epidemiologi penyelidikan, terutama di Asia Tenggara negara. Selain itu, untuk membangun link epidemiolo-gical dan untuk memahami pengenalan ND ke lokasi baru (seperti Power Tools Eropa strain-genotipe VII), itu adalah persyaratan mendasar untuk mengkarakterisasi virus di daerah mana data ini adalah kurang. Hal ini bahkan lebih penting di negara seperti Pakistan sebagai pengekspor utama burung-burung liar ke Uni Eropa, mengirim puluhan ribuan burung beo, dan burung lain ditakdirkan untuk pasar hewan peliharaan Eur-opean setiap tahun, terutama untuk Italia, Spanyol, Portugal, dan Yunani [29]. Baru-baru ini, telah diamati bahwa burung beo, kekasih dan Finch diimpor dari Pakistan ke Italia membawa penyakit Newcastle eksotis [29]. Fakta-fakta tersebut memaksakan Nasional Koleksi Power Tools strain, yang akan membuktikan berguna di masa depan epidemiologi penyelidikan.Untuk menyimpulkan, kami telah ditandai penuh gen-ome dari velogenic Power Tools dari sehat backyard unggas flock, yang merupakan milik genotipe VII dengan isolat Asia lainnya. Kami telah menyediakan bukti bagi keberadaan novel genetik sekelompok Power Tools di Pakistan dan bahwa perubahan substansial dalam situs probe M-gen didasarkan real-time PCR. Isolat ini akan sangat berharga dalam menganalisis sifat genetik APMV-1 tidak hanya di Pakistan, tetapi juga di negara-negara tetangga lainnya. Menemukan-mua-pertemuannya dalam studi ini muka data genom penuh saat ini tersedia pada APMV-1. Selain itu, kami memiliki bangunan-lished virus domestik referensi untuk studi di masa depan yang akan mengarah pada pengembangan dan pilihan sesuai strain Power Tools untuk vaksin.MetodeKoleksi virus dan isolasiDalam upaya untuk layar untuk patogen, usap kloaka, trakea dan darah sampel dikumpulkan dari ternak unggas rupanya sehat disimpan di rumah pertanian-asisten bekerja di dekat peternakan unggas. Sampel dari individu burung yang diinokulasi di lima berusia 10 hari tertentu patogen ayam gratis embrio melalui allantoic rongga. Tiga hari setelah inokulasi, allan toic cairan dipanen dan dijelaskan oleh sentrifugasi g × 4000 selama 30 menit pada 4° C. Supernatant adalah col-lected dan digunakan untuk menjalankan tes inhibisi standar penggumpalan tersebut menggunakan spesifik antisera ke galur referensi Power Tools (burung paramyxovirus tipe I). Sampel menampilkan HA titer antibodi tinggi terbagi atas saham bekerja dan disimpan pada 20 ° C. Cairan ini allantoic digunakan dalam penilaian pathogenicity dan untuk urutan analisis. Untuk deteksi genom dan karakterisasi, allantoic cairan dari setiap kawanan disimpan pada QIAcard FTA indikator empat titik (Qiagen, Hilden, Jerman), yang memelihara asam nukleat dan menonaktifkan virus. Sampel yang dikirim pada suhu ambient dari Pakistan ke Universitas Ilmu pertanian, Uppsala, Swedia, Swedia untuk diproses.Indeks intraserebral pathogenicity (ICPI)ICPI dilakukan dalam sepuluh anak ayam 1 - hari tua oleh inocula-tion dari 50 μl allantoic cairan dengan titer penggumpalan tersebut (HA) lebih dari 24 dan diencerkan 10 kali lipat dalam PBS tanpa antibiotik, disarankan [21]. Burung-burung telah dipelihara dibawah pengawasan untuk satu minggu dan ujian-ined setelah setiap 24 h. Burung-burung yang mencetak ICPI 0,7 dinyatakan sebagai lentogenic strain Power Tools dan burung-burung dengan ICPI lebih dari 1,5 dianggap velogenic. Strain Power Tools dengan nilai-nilai ICPI menengah yang ditunjuk sebagai mesogenic. Berarti kematian perhitungan waktu (MDT)Untuk MDT, cairan allantoic yang membawa viru