Altaf HUSSAlN ,WU Tian-tian ,FAN Lin-jin ,WANG Yu-long ,Farooq Khalid MUHAMMAD,JlANG Nan ,GAO Li ,Ll Kai ,GAO Yu-long ,LlU Chang-junCUl Hong-yu ,PAN QingZHANG Yan-pingAsim ASLAM,Khan MUTl-UR-REHMAN,Muhammad lmran ARSHAD,Hafiz Muhammad ABDULLAH,WANG Xiao-mei , ,Ql Xiao-le

1 Division of Avian Infectious Diseases,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,P.R.China

2 OIE Reference Laboratory for Infectious Bursal Disease,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,P.R.China

3 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses,Yangzhou 225009,P.R.China

4 Sub-campus Toba Tek Singh,University of Agriculture Faisalabad,Faisalabad 38040,Pakistan

5 Pathology Department,University of Veterinary and Animal Sciences,Lahore 54660,Pakistan

6 Institute of Microbiology,University of Agriculture Faisalabad,Faisalabad 38040,Pakistan

7 Department of Animal Sciences,Ohio State University,Columbus 43210,USA

Abstract Infectious bursal disease (IBD),caused by IBD virus (IBDV),is one of the most devastating and immunosuppressive diseases of the poultry and has been a constraint on the sustainable poultry production around the globe including Pakistan.While the disease is threatening the poultry industry,the nature of predominant strains of IBDV in Pakistan remained ill-defined.In this study,an epidemiology survey was conducted in the main chicken-farming regions of Pakistan.The batch of Pakistan IBDVs genes simultaneously covering both VP1 and VP2 were amplified,sequenced,and analyzed.The unique segmentreassortant IBDVs (vv-A/Uniq-B),carrying segment A from vvIBDV and segment B from one unique ancestor,were identified as one important type of circulating strains in Pakistan.The data also discovered the characteristic molecular features of Pakistan IBDVs,which will contribute to scientific vaccine selection and effective prevention of the disease.

Keywords:infectious bursal disease virus,circulating strains,reassortant,Pakistan

1.lntroduction

Infectious bursal disease (IBD) is one of the most important diseases of the poultry industry and poses great threats to the poultry industry worldwide (Mülleret al.2003；Qin and Zheng 2017).The IBD virus (IBDV),the causative agent of IBD,is a highly resistant virus which targets B cells within the bursa of lymphocytes (bursa),leading to immunosuppression and thus renders infected birds vulnerable to other diseases (Spackmanet al.2018；Fanet al.2019).There are two serotypes of IBDV；the serotype 1 induces clinical disease in chickens but the serotype 2 doesn't (Mülleret al.2003).Strains of IBDV belonging to serotype 1 are further classified into four types such as classical,variant,very virulent,and artificially attenuated IBDV strains (Vakhariaet al.1994).Notably,very virulent IBDV (vvIBDV) strains induce heavy morbidity and mortality in chicken and lead to heavy economic losses in the poultry industry (Sharmaet al.2000).

IBDV is non-enveloped virus with bi-segmented,linear,and double-stranded RNA genome of approximately 6.0 kb in total with 3.2 kb long segment A and 2.8 kb long segment B.Segment A encodes two open reading frames(ORF),and ORF1 encodes the non-structural protein VP5 whereas ORF2 encodes a longer precursor polyprotein (Liuet al.1994).This polyprotein is cleaved into VP2,VP3,and VP4 (Mahgoubet al.2012).VP2 and VP3 are two major structural proteins and VP2 is the major virulent determinant and host protective antigen which induces neutralizing antibodies (Qiet al.2009).VP4 is a protease,which is involved in processing precursor proteins.Segment B contains only one ORF and encodes VP1 which is an RNA dependent RNA polymerase (RdRp) and is involved in viral replication and evolution (von Einemet al.2004).

In Pakistan,poultry industry is an important industry and IBD is an important disease threatening to poultry production(Shabbiret al.2016；Khanet al.2017).While extensive vaccination programs are implemented in the country,the disease outbreaks are not uncommon.This highlights the need to characterize field strains of IBDV to know the causes of vaccine failure and to establish foundations for indigenous virus utilization for vaccine development.However,there is limited information available in Pakistan that predicts the true nature of IBDV.Restriction fragment length polymorphism (RFLP) approach has been used to identify the molecular nature of vvIBDV in Pakistan (Zahooret al.2005；Loneet al.2009).The partial sequence of VP2 gene of segment A of a few Pakistan IBDVs have been sequenced and analyzed (Shabbiret al.2016；Khanet al.2017,2018；Shafqatet al.2017；Zahidet al.2018).While these studies provide preliminary information,to our knowledge,the characterization of VP1 coded by segment B is currently lacking.Since the evolution of IBDV is determined by both segments of viral genome (Escaffreet al.2013；Yuet al.2013；Gaoet al.2014),only VP2 is insufficient to confirm the nature and dynamic of IBDV in poultry.Recently,we have determined the full-length genome of IBDV for the first time in Pakistan and the unique reassortment characteristics of two genome segments were highlighted (Hussainet al.2019),which is impossible to be discovered only by evaluating VP2 gene.In order to further ascertain the prevalence of predominant IBDV,the molecular epidemiology studies covering both segments in the main chicken farming regions in Pakistan were conducted.

2.Materials and methods

2.1.Clinical samples

From 2017 to 2018,a total of 92 bursa samples were collected from IBD suspected broilers in 29 poultry flocks from three provinces (Punjab,Sindh,and Baluchistan) and the capital territory Islamabad,which are the main chickenfarming regions in Pakistan.The 10-18% mortality was observed at these farms.The clinically diseased birds were manifested by inflammation and subsequent atrophy of the bursa,and feathers around the vent were stained with faeces containing plenty of urates.Moreover,diarrhoea,depression,and ruffled feathers were present in the region of the head and the neck.For virus detection,the bursa tissues were used to generate a homogenate in phosphate buffered saline (pH 7.2) with penicillin and streptomycin (1 g tissue:1 mL PBS).The homogenate was frozen and thawed three times before mixing thoroughly with an equal volume of chloroform and subjected to gentle agitation (20 r min-1)overnight at 4°C.The mixture was finally centrifuged at 5 000×g for 5 min at 4°C and the upper aqueous phase was transferred aseptically to sterile tubes and stored at -70°C.

2.2.Viral RNA extraction and RT-PCR

Viral RNA was extracted from the bursa homogenate using the Purelink RNA Mini Kit (Invitrogen,USA) following manufacturer instructions.Viral cDNA was synthesized using the M-MLV Reverse Transcriptase Kit (Invitrogen,Carlsbad,CA,USA).The PCR directed by PrimeSTAR HS DNA polymerase (TaKaRa Biotechnology,Dalian,China)was performed to detect IBDV in the samples as described previously (Qiet al.2015).The PCR was performed with the following protocol:preheating at 95°C for 5 min followed by 35 cycles at 95°C for 30 s,56°C for 30 s,and 72°C for 1 min,and a final extension at 72°C for 10 min.The primer pairs A628U/A1504L were used to amplify one 930-bp fragment (bp 628-1 557 of segment A) covering the hypervariable region (HVR) of the VP2 gene.The primer pairs B464U/B1718L were used to amplify one 1 255-bp fragment (bp 464-1 718 of segment B) of the VP1 gene.The primers (A628U,5′-CCTCAGCTTACCCACATC-3′；A1504L,5′-CCTTCCCCAATTGCATGG-3′；B646U,5′-AGGAGAAG CCCAATGCGT-3′；and B1718L,5′-GTCATCAATGGACC TCTC-3′) were previously designed by our laboratory(Fanet al.2019).

2.3.Sequencing

The RT-PCR products were purified with the AxyPrep DNA Gel Extraction Kit (Axygen,USA) and were sequenced by Comate Biosciences Company (Changchun,China).After being sequenced,the sequence of a 777-bp fragment(bp 547-1 323,aa 183-441) covering the HVR (aa 206-350)of VP2,and the sequence of a 1 152-bp fragment (bp 400-1 551,aa 134-517) of VP1,were determined and submitted to GenBank.

2.4.Sequence analyses

A total of 36 IBDV isolates (one or two isolates per flock) were selected for the sequence analysis of both VP1 and VP2 (Table 1).Alignment and phylogenetic analyses based on the nucleotide (nt) or amino acid (aa)sequences were performed using the Clustal W (version 1.8) (Thompsonet al.1997) and the MEGA (version 3.1) (Kumaret al.2004),and the confidence levels were assessed using 1 000 bootstrap replications.The types and the GenBank accession numbers of reference IBDVs used in this study are as follows:vvIBDV,UK661 (UK) (NC-004178,NC-004179),D6948 (Netherlands) (AF240686,AF240687),HK46 (China) (AF092943,AF092944),YS07(China) (FJ695138,FJ695139),BD399 (Bangladesh)(AF362776,AF362770),OKYM (Japan) (D49706,D49707)；attenuated strains,CEF94 (Netherlands) (AF133904,AF133905),CT (France) (AJ310185,AJ310186),CU-1(Germany) (X16107,AF362775),D78 (USA) (AF499929,AF499930),Gt (China) (DQ403248,DQ403249),HZ2(China) (AF321054,AF493979),JD1 (China) (AF321055,AY103464),NB (China) (AY319768,AY654284),P2(Germany) (X84034,X84035)；classic strain,IM (USA)(AY029166,Y029165)；variant strains GLS (USA)(AY368653,AY368654),Variant E (USA) (AF133904,AF133905),9109 (USA) (AY462027,AY459321)；Serotype II,23/82 (Germany) (AF362773,AF362774),OH (Canada)(U30818,U30819)；and reassortment strains (vv-A/Uniq-B),HLJ0504 (China) (GQ451330,GQ451331),Gx (China)(AY444873,AY705393),Harbin-1 (China) (AF092171,AF454945),02015.1 (Venezuela) (AJ879932,AJ880090),West Bengal/HBL-07-15 (India) (KT630845,KT630853),West Bengal/HBL-07-15-b (India) (KT630847,KT630855).The Pakistan representative IBDVs isolated previously with published sequence of appropriate length are as follows:PK2 (MF996499,MF996500),IBDV-PK-1 (KT281984),PUN-PAK-007-2014 (KY484079),PUN-PAK-026-2015(KY484077),PUN-PAK-030-2015 (KY484078),PUNPAK-037-2016 (KY000833),PUN-PAK-038-2015(KY484080),PUN-PAK-044-2015 (KY484081),PUNPAK-045-2015 (KY484082),PUN-PAK-046-2016(KY484083),PUN-PAK-051-2016 (KY484084),and PUNPAK-126-2016 (KY484085).To be mentioned,because of the short length of gene sequence of West Bengal/HBL-07-15 strain and West Bengal/HBL-07-15-b strain submitted to GenBank,sequence analysis about these two India strains were based on a 435-bp fragment (bp 616-1 050,aa 206-350) covering the HVR of VP2 and a 411-bp fragment (bp 400-810,aa 134-270) of VP1.

3.Results

3.1.lBDV detection

RT-PCR results showed that 87% (80/92) bursa tissues were positive for IBDV.The 36 representative IBDV isolates covered 29 broiler flocks in three provinces and the capital territory of Pakistan were selected to perform sequence analysis for both partial fragments of VP1 and VP2 (Table 1).The gene sequences of both VP1 and VP2 of all 36 Pakistan strains were submitted to GenBank and the accession numbers are listed in Table 1.

3.2.Molecular characterization of VP2 gene of Pakistan lBDV isolates

Sequence similarity analysis of the representative part of VP2 showed that all 36 Pakistan strains isolated in this study shared identity of 99-100% (nt) and 99.2-100% (aa) with each other.Meanwhile,they shared high identity (nt,98.5-100%；aa,98.8-100%) with other Pakistan strains isolated previously listed in Section 2.4.Besides,the Pakistan strains shared 94.6-96.3% (nt) and 98.1-99.2% (aa) identity with vvIBDV but only 91.0-92.5% (nt) and 93.8-97.7% (aa) with non-vvIBDV strains (including classical,variant,attenuated strains).In addition,based on a 435-bp fragment of VP2,Pakistan IBDV strains showed identity of 93.6-94.7% (nt) and 97.3-98.6% (aa) with India strain of West Bengal/HBL-07-15.The identity comparison results of VP2 fragments of IBDV strains isolated in this study with other representative strains of each type were shown in Table 2.Furthermore,sequence alignment of VP2 showed that eight characteristic aa residues of vvIBDV (222A,242I,253Q,256I,279D,284A,299S,and 330S) (Luet al.2015) also presented in Pakistan IBDVs.Additionally,two distinct amino acid residues (210N and 384I)were observed in Pakistan IBDVs,which are different from vvIBDV and non-vvIBDV.

3.3.Molecular characterization of VP1 gene of Pakistan lBDV isolates

In sequence homology analysis of the representative part of VP1,all 36 Pakistan IBDV strains shared identity of 98.9-100% (nt) and 98.4-100% (aa) with each other.Pakistan IBDV strains showed similar identity with vvIBDV(nt,87.6-88.6%；aa,96.1-97.9%) and non-vvIBDV (nt,88.1-90.5%；aa,96.4-98.4%).VP1 of Pakistan IBDV strains had comparatively higher identity of 89.9-91.3% (nt) and 97.4-99.0% (aa) with HLJ0504-like strains,which are theunique reassortment strains with segment A from vvIBDV and segment B from unique ancestor (vv-A/Uniq-B) (Qiet al.2011；Heet al.2014；Hussainet al.2019).In addition,based on a 411-bp fragment of VP1,Pakistan IBDV strains showed identity of 98.5-99.3% (nt) and 98.6-100% (aa)with India strain of West Bengal/HBL-07-15.The identity comparison results of VP1 fragments of IBDV strains isolated in this study with other representative strains of each type were shown in Table 2.In sequence alignment of the representative part VP1,Pakistan IBDVs had three characteristic aa residues of vvIBDV (287A,508K,and 511S)and four characteristic aa residues of non-vvIBDV (146E,147G,242D,390L,and 392E).In addition,Pakistan IBDVs had three distinct aa residues (141I,143D,and 145S),which were same as India strains of West Bengal/HBL-07-15 and West Bengal/HBL-07-15-b.

Table 2 Identity comparison of VP1 or VP2 fragments of infectious bursal disease virus (IBDV) strains isolated in this study with other representative strains

3.4.Phylogeny analysis of Pakistan lBDV isolates

The phylogenetic tree based on the nucleotide sequences of VP2,except serotype II strains (OH and 23/82),was distinctly divided into two major branches,vvIBDV and non-vvIBDV.All 36 Pakistan IBDV strains isolated in the study belonged to vvIBDV branch.In vvIBDV branch,all 36 Pakistan IBDV strains isolated in the study and other 12 other Pakistan representative IBDV strains formed one subgroup (Fig.1-A).In the phylogenetic tree based on the nucleotide sequences of VP1,serotype I strains revealed three distinct branches,however,all Pakistan IBDV strains were branched out of the vvIBDV branch and formed a unique cluster with HLJ0504-like strains,including HLJ0504,Gx,Harbin-1,02015.1,and PK2 (Fig.1-B).In addition,the phylogenetic tree based on the nucleotide sequences of VP2(435-bp fragment) and VP1 (411-bp fragment) showed that India strains of West Bengal/HBL-07-15 and West Bengal/HBL-07-15-b were located in the same cluster with Pakistan IBDV strains (Appendix A).

4.Discussion

IBD appeared to be an important disease and has threatened the poultry industry in Pakistan for the last fifty years (Shafqatet al.2017；Shahet al.2018).IBD caused major losses to poultry farmers due to unawareness of controlling measures in Pakistan (Shafqatet al.2017).Although gene sequencing has been used to analyze Pakistan IBDV strains recently(Shabbiret al.2016),only a few sequences of VP2 while even no VP1 sequence of Pakistan IBDVs were available in GenBank.Recently,the unique IBDV recombined by two genome segments in Pakistan was identified for the first time by our team (Hussainet al.2019),which discovered the novel epidemic characteristics of IBDV prevailing in Pakistan.To further evaluate the epidemic status of the unique recombinant IBDV,IBDVs were detected in 29 poultry flocks,which located in three provinces (Punjab,Sindh,and Baluchistan) and the capital territory Islamabad of Pakistan.Among them,Punjab Province is the most populous province and it is a very important chicken-farming region in Pakistan.This epidemiologic study was a passive surveillance with an active research purpose.We will further enlarge the scope of sample collection.Representative fragments of both VP1 and VP2 of 36 IBDVs were sequenced,analyzed,and submitted to GenBank,which are the first batch of IBDV sequences simultaneously covering both VP1 and VP2 of Pakistan strains in GenBank while only VP2 genes of Pakistan IBDVs were available in GenBank before.

All 36 Pakistan IBDVs isolated in this study shared close identity of both VP1 and VP2 with each other.Compared with VP2 of the other 12 Pakistan representative IBDVs isolated previously,Pakistan IBDVs showed close evolutionary relationship.For VP2,Pakistan IBDVs showed higher nucleotide identity with vvIBDV (above 94.6%) than non-vvIBDV (below 92.5%),which means VP2 of Pakistan IBDVs belong to vvIBDV.Interestingly,VP1 of Pakistan IBDVs showed similar lower evolutionary identity of nucleotide with vvIBDV (87.6-88.6%) and nonvvIBDV (88.1-90.5%).It seemed that Pakistan IBDVs exhibited different genetic relatives for segments A and B.Furthermore,the phylogenetic trees verified the results of sequence alignment.Segment A-coded VP2 of Pakistan strains was clustered into one branch with vvIBDV,while VP1 coded by segment B branched out of the vvIBDV branch and formed a unique branch with HLJ0504-like strains.It is well known that HLJ0504-like strains,including HLJ0504,Gx,Harbin-1,02015.1,and PK2,belong to unique segment-reassortant strains (vv-A/Uniq-B) of which segment A originated from vvIBDV but segment B derived from a unique ancestor (Honet al.2008；Heet al.2014；Qiet al.2015；Pikulaet al.2018；Hussainet al.2019).Therefore,Pakistan IBDVs isolated in this study are unique segmentreassortant IBDVs (vv-A/Uniq-B).

It was reported that vv-A/Uniq-B IBDVs (HLJ0504-like strains) were the popular strains in China (Heet al.2014；Qiet al.2015).Recently,this type of IBDVs were also detected in Venezuela (Le Nouenet al.2006),Nigeria(Nwagboet al.2016),India (Patelet al.2016),and Algeria(Abedet al.2018).All 36 Pakistan IBDVs isolated in this study have high identity of VP2 with other Pakistan strains isolated previously listed in Section 2.4,but their homology relations of VP1 are unknown because no VP1 sequences of early Pakistan strains are available.This study showed that the unique segment-reassortant IBDVs (vv-A/Uniq-B)are one important type of circulating strains in Pakistan at least since 2017.

Fig.1 Phylogenetic tree analysis of the nucleotide (nt) sequences of the representative fragments of VP2 (nt 547-1 323) (A)and VP1 (nt 400-1 551) (B).The trees were generated by the neighbor-joining method using MEGA (version 3.1).The Pakistan infectious bursal disease virus (IBDV) strains detected in this study are highlighted with a solid circle.

In vv-A/Uniq-B group,Pakistan IBDVs had specific characteristics.Pakistan IBDVs were clustered into a distinct subgroup,which showed nucleotide identity of 95.0-96.3% (VP2) and 89.9-91.2% (VP1) with other vv-A/Uniq-B IBDVs except India strains mentioned above.Distinct amino acid residues in VP2 (210N and 384I) and in VP1 (141I,143D,and 145S) existed in Pakistan IBDVs.VP1 of IBDV includes three domains:N-terminal domain(aa 1-167),the central polymerase domain (aa 168-658),and C-terminal domain (aa 659-878) (Panet al.2007).Pakistan IBDVs showed recombinant characters between very virulent and attenuated strains in the N-terminal domain and the central polymerase domain.The N-terminal domain(aa 1-67) contains the putative self-guanylylation site of RdRp and is responsible for protein priming of IBDV.The amino acid triplets at positions 145/146/147 of VP1 are important virulent sites through influencing the RdRp (Panet al.2007).It has been reported that there were three types of the triplet,TDN,TEG,and NEG,which were the characteristic of very virulent,HLJ0504-like,and attenuated IBDV,respectively (Gaoet al.2014).Interestingly,VP1 of almost all Pakistan IBDVs has the fourth kind of triplet (SEG),as the same with a few India strains (Patelet al.2016) and Uruguay strains (Hernandezet al.2015).Besides,PK28 strain showed the fifth kind of triplet (SGG).The central domain (aa 168-658),containing all of the structural motifs of RdRp,is suggested to be involved in nucleotide recognition and binding (Panet al.2007).Pakistan IBDVs had the same residues with vvIBDV (287A,508K,and 511S)and non-vvIBDV (242D,390L,and 392E) in the central polymerase,of which the biological significances can be further explored using the reverse genetics systems (Mundt and Vakharia 1996；Qiet al.2007).

Since the unique segment-reassortant IBDVs (vv-A/Uniq-B) are also circulating in neighboring countries of Pakistan including China and India,it is interesting to explore the relations among them.The gene sequences length of representative strains of India reassortant IBDVs(West Bengal/HBL-07-15 and West Bengal/HBL-07-15-b)submitted to GenBank is short,so we shortened our sequences to do the comparison.Sequence analysis results based on the nucleotide sequences of a 411-bp fragment of VP1 and a 435-bp fragment of VP2 showed that Pakistan IBDVs have high identity of VP1 (98.5-99.3%) but relatively low identity of VP2 (93.6-95.6%) with India representative strains.In addition,Pakistan IBDVs showed low identity of VP1 (90.3-91.3%) but relatively high identity of VP2(95.0-96.1%) with HLJ0504-like strains of China.Totally,Pakistan strains,India strains,and China strains mentioned above belonged to the unique segment-reassortant IBDVs(vv-A/Uniq-B) but they had obvious genetic distance.The origin and travel path of the vv-A/Uniq-B IBDVs deserves further study with more epidemiological data and systematic experiments in the future.

Usually,co-evolution of both genome segments is a major evolutionary rule in IBDV (Le Nouenet al.2006).However,recently,six types of segment-reassortant IBDV were discovered:vv-A/att-B (Sunet al.2003；Chenet al.2012；Kasangaet al.2013；Luet al.2015；Senthilkumaret al.2016；Leeet al.2017；Pikulaet al.2018),att-A/vv-B(Weiet al.2006；Cuiet al.2013),vv-A/c-B (Fernandeset al.2012；Gallardoet al.2014),vv-A/var-B (Le Nouenet al.2006),vv-A/II-B (Jackwood and Sommer-Wagner 2011；Soubieset al.2017),and vv-A/Uniq-B (Gaoet al.2007；Honet al.2008；Xiaet al.2008；Qiet al.2011；Heet al.2014；Patelet al.2016；Hussainet al.2019) (att,attenuated strain；c,classic strain；var,variant strain；II,serotype II strain).A few complicated factors including vaccinations,selection pressure,host immunity,and environment might be involved in genome reassortment,which have not been verified experimentally (Honet al.2006；Le Nouenet al.2006).As one necessary gene in IBDV evolution (Honet al.2006),the origin of segment B of vv-A/Uniq-B strains is still unknown.It is speculated that the unique B might exist in other reservoir hosts like duck,goose,sparrow,or other wild birds (Fagbohunet al.2000；Jackwoodet al.2005；Wanget al.2007).

5.Conclusion

The unique segment-reassortant IBDVs (vv-A/Uniq-B),carrying segment A from vvIBDV and segment B from one unique ancestor,were identified as one important type of circulating strains in Pakistan.The batch of Pakistan IBDV sequences simultaneously covering both VP1 and VP2 were analyzed in whole,which scientifically discovered the real epidemic characteristics and situation of IBDV in Pakistan.The present study will contribute to scientific vaccine selection and effective prevention of the disease.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2016YFE0203200,2017YFD0500704),the Heilongjiang Province Foundation for the National Key Research and Development Program of China (GX18B011),the National Natural Science Foundation of China (31430087),and the earmarked fund for the China Agriculture Research System (CARS-41-G15).

Appendixassociated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm