riewarticle - hindawimar 23, 2018 · biomedresearchinternational hf969184.1 chicke/i or c...

Review ArticleDiagnostic and Vaccination Approaches for Newcastle DiseaseVirus in Poultry The Current and Emerging Perspectives

Muhammad Bashir Bello 12 Khatijah Yusoff 13 Aini Ideris 14

Mohd Hair-Bejo 15 Ben P H Peeters6 and Abdul Rahman Omar 15

1Laboratory of Vaccines and Immunotherapeutics Institute of Bioscience Universiti Putra Malaysia43400 Serdang Selangor Malaysia

2Department of Veterinary Microbiology Faculty of Veterinary Medicine Usmanu Danfodiyo University Sokoto Nigeria3Department of Microbiology Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia Selangor Malaysia4Department of Veterinary Clinical Services Faculty of Veterinary Medicine Universiti Putra Malaysia Selangor Malaysia5Department of Veterinary Pathology andMicrobiology Faculty of VeterinaryMedicine Universiti PutraMalaysia SelangorMalaysia6Department of Virology Wageningen Bioveterinary Research Lelystad Netherlands

Correspondence should be addressed to Abdul Rahman Omar aroupmedumy

Received 23 March 2018 Revised 25 June 2018 Accepted 16 July 2018 Published 5 August 2018

Academic Editor Xiaofeng Fan

Copyright copy 2018 MuhammadBashir Bello et alThis is an open access article distributed under theCreativeCommons AttributionLicensewhichpermits unrestricteduse distribution and reproduction in anymedium provided the original work is properly cited

Newcastle disease (ND) is one of the most devastating diseases that considerably cripple the global poultry industry Because of itsenormous socioeconomic importance and potential to rapidly spread to naıve birds in the vicinity ND is included among the list ofavian diseases that must be notified to the OIE immediately upon recognition Currently virus isolation followed by its serologicalor molecular identification is regarded as the gold standard method of ND diagnosis However this method is generally slow andrequires specialised laboratory with biosafety containment facilities making it of little relevance under epidemic situations whererapid diagnosis is seriously neededThus molecular based diagnostics have evolved to overcome some of these difficulties but theextensive genetic diversity of the virus ensures that isolates with mutations at the primerprobe binding sites escape detection usingthese assays This diagnostic dilemma leads to the emergence of cutting-edge technologies such as next-generation sequencing(NGS) which have so far proven to be promising in terms of rapid sensitive and accurate recognition of virulent Newcastledisease virus (NDV) isolates even in mixed infections As regards disease control strategies conventional ND vaccines have stoodthe test of time by demonstrating track record of protective efficacy in the last 60 years However these vaccines are unable toblock the replication and shedding of most of the currently circulating phylogenetically divergent virulent NDV isolates Hencerationally designed vaccines targeting the prevailing genotypes the so-called genotype-matched vaccines are highly needed toovercome these vaccination related challenges Among the recently evolving technologies for the development of genotype-matchedvaccines reverse genetics-based live attenuated vaccines obviously appeared to be the most promising candidates In this reviewa comprehensive description of the current and emerging trends in the detection identification and control of ND in poultry areprovided The strengths and weaknesses of each of those techniques are also emphasised

1 Introduction

Newcastle disease (ND) is one of the most important aviandiseases that significantly affect poultry production all overthe world [1] From its first official report in 1926 at NewcastleUpon Tyne in England to date ND has accounted fortremendous economic losses through numerous epidemicsassociated with high mortality high morbidity and many

other production related losses Consequently the WorldOrganisation for Animal Health (OIE) has included it amongthe list of diseases that require immediate notification uponrecognition [2] The aetiology of the disease is Newcastledisease virus (NDV) a negative stranded RNA virus whose152 kb nonsegmented genome is organised into six genesencoding six structural proteins namely NP PM F HN andL as well as two nonstructural proteins V andW [3] Among

HindawiBioMed Research InternationalVolume 2018 Article ID 7278459 18 pageshttpsdoiorg10115520187278459

2 BioMed Research International

these proteins the F is generally considered to be amolecularmarker of NDV virulence According to the OIE virulentNDV strains are diagnosed as those possessing multiple basicamino acid residues (arginine and lysine) at the F proteincleavage site located between amino acid positions 112-116and a phenylalanine residue at position 117 On the otherhand isolates of low virulence are considered to be those withmonobasic F cleavage site and a leucine residue at position117 [2] Unfortunately this is not a universal rule of thumbas some NDV strains adapted in wild birds such as pigeonsand doves have been shown to be minimally pathogenicdespite possessing the so-called polybasic F cleavage site [45] Furthermore some of these pigeon paramyxoviruses havebeen reported to manifest a dramatic increase in virulencefollowing a few passages in chicken without the accompany-ing nucleotide changes in the entire F coding region [6 7] Inaddition swapping the F genes of avirulent pigeon adaptedNDV with that of a virulent NDV strain or vice versa didnot lead to the change in the virulence of the generatedchimeric viruses These observations altogether signify thatother factors independent of the F cleavage site are importantin determining the virulence of NDV in chicken Indeedthe HN and L proteins have recently been proven to bedirectly associated with NDV virulence [8 9] Hence thebiggest diagnostic dilemma is whether targeted evaluationof F cleavage site is still a reliable molecular indicator ofNDV virulence or complete genome analysis is required toadequately predict the virulence potential of NDV isolates

Epidemiological evidences indicate that NDV is con-stantly evolving and its isolates are so far classified intomore than eighteen phylogenetically distinct genotypes [14]Regardless of their genetic variability however all NDVisolates are serologically grouped into a single serotype calledavian paramyxovirus type-1 [15] This implies that immunityinduced by any NDV strain should be able to cross-protectagainst challenge with any other virulent strain because ofthe fairly similar antigenic properties shared by all NDVisolates Indeed for over sixty years both live attenuatedand inactivated NDV vaccines have been extensively used tocurb the economic menace of ND across the globe [16] Inparticular the live vaccines are known for their track recordof high efficacy due to their ability to efficiently replicateand induce a robust immune response following a singleadministration They are also suitable formass application viaspray or drinking water [17] Unfortunately they are unableto block the replication of the heterologous virulent NDVdespite protecting against overt clinical disease [18] As aresult birds vaccinated with those vaccines may serve asreservoirs for virulentNDV shedding a substantial amount ofthe infectious virus into the environment leading to potentialoutbreaks among the nonprotected in-contact birds [19]These shortcomings of the conventional vaccines collectivelycall for the need to improve the current vaccination strategiesagainst the prevalent NDV strains across the globe

Recently the global poultry industry has witnessed anupsurge in the emergence of cutting-edge techniques for theidentification and control of virulent NDV infection Thesestate-of-the-art technologies have no doubt demonstrated thepotentials to overcome the loopholes of their conventional

counterparts Here we comprehensively review the currentand next-generation diagnostic and vaccination strategies forNDV with special emphasis on the strengths and weaknessesof each of those techniques

2 Etiology

21 Classification of NDV Isolates of NDV are membersof the genus Avulavirus in the family Paramyxoviridae Atpresent a total of 15 avian paramyxovirus serotypes (APMV-1to APMV-15) have been identified in different species of wildand domesticated birds where they may be associated withrespiratory disease and a drastic reduction in egg production[20] Members of APMV-1 made up of NDV isolates are themost economically important and are genetically classifiedinto two broad groups class I and class II Whereas membersof class I isolates are grouped into only one genotype isolatesbelonging to class II are further subdivided into genotypes I-XVIII which are all predicted to be virulent in chicken exceptsome isolates in genotypes I II and X [21 22]

22 Molecular Biology of NDV The genetic material ofNDV is a negative stranded nonsegmented RNA that strictlyadheres to the rule of six (genome size divisible by six)having a total size of 15186 15192 or 15198 bp [23] Thegenome houses six genes namely nucleocapsid protein (NP)phosphoprotein (P) matrix protein (M) fusion protein (F)hemagglutinin-neuraminidase protein (HN) and the largeprotein (L) Apart from the P gene which can be transcribedinto three different mRNAs encoding one structural (P)and two nonstructural (V and W) proteins [24] all otherviral genes are monocistronic encoding a single structuralprotein The 31015840 and 51015840 ends of the viral genome constitute theleader and trailer regions which accommodate the regulatorysignals for virus transcription and replication [25]

Morphologically NDV appears to be a pleomorphicenveloped particle with projections of F and HN spike gly-coproteins (Figure 1) that participate in the initiation of virusinfectious cycle (Sachin et al 2011) Immediately beneath theviral envelope is the M protein which is known to maintainthe shape of the virus and assist in the packaging and releaseof the newly assembled viruses [26] The other three proteinsare intimately associated with the viral genome and areknown to perform replication related functions In particularthe NP precisely covers the entire viral RNA to form theribonucleoprotein (RNP) which is the minimum templaterequired for virus replication and mRNA biosynthesis [27]The L protein is the RNA dependent RNA polymerase thatserves as the viral replicase and transcriptase during theinfectious cycle while the P protein is the cofactor of thepolymerase [8]

23 Epidemiology and Emergence of the Virulent NDV Inthe last nine decades four major economically devastatingNDV panzootics have occurred The first one which begansimultaneously in Asia and Europe around the mid-1920swas spreading to the rest of the world rather slowly andtook about twenty years to become fully established [28]Thesecond pandemic however became full pledged within four

BioMed Research International 3

NP

P

M

F

HN

L

NP P M F HN L

Figure 1 Structural features of Newcastle disease virus AMorphology of the virion showing the locations of the viral proteinsNP P and L proteins associate with the RNA genome to form RNPwhile the M F and HN are membrane associated B Arrangementof the genes in the viral genome

years [29] presumably due to the increased commercialisa-tion of poultry industry globally as well as the enhancedinternational trade of captive cage birds which were shownto be the reservoirs of virulent NDV in different parts of theworld [30 31] The third panzootic believed to be caused bygenotype VI isolates in the mid-1980s occurred among theracing pigeons but eventually affected several bird speciesand became difficult to control due to the relative lack ofabsolute control in the racing pigeon husbandry [32] Finallythe fourth pandemic which is currently ongoing is believedto have started from the late 1980s and has been linkedto several economic losses in a vast number of countriesacross the South East Asia Middle East Europe Africa andAmerica [33ndash35] This particular panzootic has been shownto be caused by the genotype VII group of NDV whichcurrently constitute the most rapidly evolving strains of thevirus (Figure 2) Given the recent expansion in the geographicdistribution and host range of some of these genotype VIIstrains the fifth panzootic is strongly anticipated in the nearfuture [36]

The emergence of virulent NDV isolates is certainly aserious concern in the global poultry industry Apparentlylentogenic strains predominantly harboured by wild birds[37] are among the major reservoirs for the emergence ofthe virulent NDV in poultry Through ecological contactinterfaces these lentogenic viruses have been shown to beeasily be transmitted fromwild birds to domesticated poultrywhere they are silently maintained without causing anyclinical disease [38] However continuous multiplication oflentogenic NDV in chicken is a potential risk factor for theemergence of the virulent NDV Meng et al [39] serially pas-saged a duck origin lentogenicNDVstrain 10 times in chickenair sacs and showed that the virus dramatically acquiredvirulence as measured by standard pathogenicity assessmentindices When ultra-deep sequencing of the partial genome

encompassing the F cleavage site of the passaged virus wasperformed it was revealed that the proportion of the virulentNDV variants within the pool of the viral quasispeciesgradually accumulated as the passage number increased [39]Similarly vaccine derived NDV strains used in domesticatedchicken have been reported to occur in wild birds [37] whichfurther provides evidence of virus exchange between thewild and domesticated avian species at ecological contactpoints Thus the continuous circulation and maintenance oflentogenic NDV between the wild and domesticated birdsconstitute a huge threat for the emergence of virulent NDVstrains of huge economic consequences

24 Pathotypes and Pathotyping of NDV All virulent NDVisolates require immediate notification to the OIE [40]Hence pathotype identification is necessary for a completediagnosis of NDV in poultry Using molecular based assaysthe predicted amino acid sequence of the F cleavage sitecan be used to classify NDV isolates into virulent andavirulent strains [41] Isolates with polybasic F cleavage siteand phenyl alanine at position 117 are considered virulentwhile those with monobasic F cleavage site and leucineresidue at position 117 are classified as avirulent Based onsome in vivopathogenicity assessment tests NDV isolates canalso be classified into velogenic (very virulent) mesogenic(moderately virulent) and lentogenic (avirulent) pathotypes[42] The commonest of such tests is the mean death time(MDT) performed in 9-10-day-old embryonated chickeneggs which is the average time (in hours) taken for the meanlethal inoculum of the virus to kill all the inoculated embry-onated eggs [43] As a general rule isolates are diagnosedas velogenic when their MDT is 40-60 hours or mesogenicif they have MDT values of 60-90 hours Lentogenic strainsnormally have MDT values of greater than 90 hours [44] Atpresent the most widespread NDV pathotyping tool is theintracerebral pathogenicity index (ICPI) performed in 1-day-old SPF chicks [45] It is normally scored between 0 and 2with virulent strains having values from 13 to 20 while thevalues for mesogenic strains range from 07 to 13 Lentogenicisolates generally have ICPI values of 00-07 [46] Finallya useful but less popular virulence determination test is theintravenous pathogenicity index (IVPI) performed in 4-6-week-old SPF chicken Its scores range from 0 to 3 and itis normally proportional to the virulence of the virus [46]Thus isolates with IVPI value of 00 are lentogenic whilemesogenic strains have values between 00 and 05 and thevalues for velogenic strains range between 05 and 30 Insummary for an isolate to qualify as notifiable to the OIEit has to be classified as virulent by possessing at least one ofthe following poly basic F cleavage site MDT value of 40-60hours and ICPI value of gt 13 or IVPI gt 05

3 Diagnosis

31 Diagnostic Dilemma Poultry respiratory pathogens suchas avian influenza infectious bronchitis and infectious laryn-gotracheitis viruses are all considered differential diagnosesthat can easily be confused with NDV based on their clinicalpresentation [47] In fact some avian paramyxoviruses such


HF9691841 chickenIvory CoastCIV08-1032007 JF9663851 2008_Mali_ML007_08 FJ7724841 chicken-3490-147-Cameroon-2008

XVII

HF9692181 chickenIvory CoastCIV08-0422007 HG3266001 village weaverIvory CoastCIV08-0322006

XVIII

JX5462451 NDVchickenBenin463MT2009 HF9691451 chickenNigeriaNIE09-20142009 HF9691491 chickenNigeriaNIE09-20412009

XIV

GU5859051 strain chickenSweden97 KJ5775851 NDVChickenBareilly0110

XIII

HQ6972541 chickenBanjarmasin01010 KF1133391 NDV isolate 752 KT3555951 NDV isolate IBS02513 KX4969621 wild pigeonPakistanLahore20A9962015

VII

JN8003061 poultryPeru1918-032008 KR7326141 NDVpeacockPeru2011

XII

KU5275591 PigeonChinaJilinDH092015 JX4865521 strain piCHLLN110713 KJ7823761 pigeonCHNM07072011

VI

AY5629861 isolate anhingaUS(Fl)4408393 AY5629901 mixed speciesUSLargo71

V

JX0120962 strain AF2240-I MF2850771 isolate HR09

VIII

JX1869971 chickenDominican Republic867-22008 JX9152421 chickenDominicanRepublic28138-41986

XVI

AY7414041 strain Herts33 EU2939141 strain Italien

IV

HQ2666021 strain MG_725_08 HQ2666031 strain MG_1992

XI

FJ4301601 JS905Go JF9505091 NDV strain Mukteswar

III

FJ4363031 strain ZJ186Ch KR0148141 isolate DuckCHGDYF14

IX

GQ2883911 mottled duckUS(TX)01-1302001 JN8721711 TurkeyMinnesota17531-32010

X

KT4459011 strain Komarov AY2890021 turkeyUSAVGGA89

DQ1952651 strain LasotaII

JX1930811 duckChinaGuangxi202010 KX8577101 Blue-winged tealUSATXAI14-37112014

KX8577081 MallardUSAMNAI14-33102014I

Class II

KC5034431 northern pintailAK44420-0882008 EF5648231 duckUS(OH)04-6122004 FJ5975801 strain D_ZJ_1_02

EF5648331 Canada gooseUS(OH)87-781987 EU4934512 APMV-1TealFinland1210406

Class I100

100

100

77100

100

100

100

99100

100

100

100

100100

100

100

100

100

100

100

100

96

78

92

85

73

100

92

84

98

005Figure 2Phylogenetic relationships ofNewcastle disease virus genotypes (shown in roman numbers) using the complete F gene codingsequences (1662bp) Red coloured taxon represents the group currently causing the wave of the fourth ND pandemic Taxon containing thecurrent vaccine strains is shownwith green colourThe evolutionary historywas inferred using theNeighbor-Joiningmethod [10]The optimaltree with the sum of branch length = 160126925 is shown The percentage of replicate trees in which the associated taxa clustered togetherin the bootstrap test (1000 replicates) is shown next to the branches [11] The tree is drawn to scale with branch lengths in the same unitsas those of the evolutionary distances used to infer the phylogenetic tree The evolutionary distances were computed using the MaximumComposite Likelihood method [12] and are in the units of the number of base substitutions per site The analysis involved 46 nucleotidesequences Codon positions included were 1st+2nd+3rd+noncoding All positions containing gaps and missing data were eliminated Therewere a total of 1656 positions in the final dataset Evolutionary analyses were conducted in MEGA6 [13]


APMV-3 and 7 may even cross react with NDV in routineserological diagnosis [48] Thus it is important to rapidlyidentify the strains of NDV and differentiate them fromotherclosely related pathogens so that correct intervention fordisease control can be applied As a pathogen of several avianhosts NDV demonstrates a wide spectrum of virulence thatranges from highly fatal to subclinical disease This virulencespectrum is largely controlled by the genetic makeup ofboth the virus and avian hosts Wild birds for instancecan maintain highly virulent NDV without showing obviousclinical disease symptoms [49] Backyard chicken geeseand ducks which are naturally less susceptible to virulentNDV are also incriminated in maintaining the virus in theenvironment [50] Furthermore exotic pet birds kept inclose proximity to commercial poultry have been shownto harbour virulent NDV strains that are genetically highlyrelated to those obtained in the commercial farms [51] Thisoccurrence of virulent NDV in apparently healthy wild andbackyard poultry not only constitutes a huge diagnosticchallenge but also represents a big biosecurity threat to thecommercial poultry industry

The OIE recommended in vivo tests used to identifyvirulent NDV isolates have certainly proven to be usefulin ND diagnosis However they often give contradictoryresults An isolate classified as mesogenic using the MDTmay turn out to be velogenic based on the ICPI or IVPI tests[52] Furthermore pathotyping of NDV isolates obtainedfrom species other than chicken may not yield very accurateresults until the isolates are passaged in chicken or chickenembryonated eggs More so the ICPI test regarded as themost robust OIE recommended pathogenicity test may notperfectly depict the real virulence of the virus because itutilises a route that does not represent the natural route ofNDV infection [6 7] These diagnostic dilemma leads to theconclusion that the best indicator of NDV virulence in aparticular avian species should be the experimental infectionof a statistically significant number (ge10) of young and adultbirds with a standard amount of the virus inoculum vianatural routes [53] There is therefore the need to improvethe current pathotyping systems so that virulent NDV canbe rapidly and accurately identified and contained beforedevastating loss of poultry is incurred

An important virus related factor that contributes to thedilemma in the diagnosis of virulent NDV is the aminoacid composition of the F cleavage site According to theOIE virulent NDV isolates are identified by the possessionof multiple basic amino acids at the F cleavage site whichcan be cleaved by all furin-like intracellular proteases ubiq-uitously distributed all over the body [2] On the other handavirulent isolates are those possessing monobasic F cleavagesite cleavable by extracellular trypsin like proteases foundlargely in the gastrointestinal and respiratory systems Thismakes the molecular prediction of the virulence potentialof NDV to be solely based on the chemistry of F cleavagesite [54] Contrastingly emerging evidences indicate thatother NDV genes might considerably contribute to the viralvirulence In one study a single passage of a recombinantNDV strain LaSota encoding a velogenic F cleavage site inpigeon leads to a dramatic increase in ICPI from 13 to 17

without any change in the entire nucleotide sequence of theF gene [55] In another study pigeon derived NDV strainsexpressing the velogenic F cleavage site were shown to becompletely avirulent in chicken especially at first passageHowever after several passages the viruses became highlyvirulent even though no obvious nucleotide substitution inthe entire F gene of the viruswas observed [56]This indicatesthat virulence of NDV is multigenic and that other factorsindependent of the F cleavage site are crucial in determiningthe virulence of the virusThus the dilemma in the diagnosisof virulent NDV demands a revisit and improvement ofthe current pathotyping tools so that the virulence of NDVisolates can be more accurately predicted

32 Clinical Diagnosis

321 Clinicopathological Features On the basis of the clinicaland pathologic manifestations five different forms of NDare recognised [57] The severest form is the velogenicviscerotrophic ND (VVND) characterised by mortality andmorbidity rates approaching 100 [58] It is associated withconjunctivitis nasal discharges dyspnoea diarrhoea ruffledfeathers prostration tremors and paralysis At postmortemulcerative haemorrhages may be observed throughout thedigestive tract especially at the proventriculus-gizzard junc-tion and in the caecal tonsils [42] Necrotic foci may also bealso observed in some internal organs such as the spleen liverand gut associated lymphoid tissue (GALT) Histologicallythe spleen and the Peyerrsquos patches showmicroscopic evidenceof necrosis and haemorrhage In the nervous system apartfrom perivascular cuffing no neurological lesions due toVVND are observed even among birds that died showingneurological symptoms [53]

Another form of the disease is velogenic neutrotropic ND(VNND) characterised by neurological and some respiratoryclinical signs with no gastrointestinal involvement Typi-cally the affected birds manifest opisthotonus tremors headtwisting and paralysis Gross lesions are often absent evenamong birds that died showing typical symptoms Howeverat histology necrosis of Purkinje fibres as well as perivas-cular cuffing are highly encountered [59] Mesogenic ND(MND) is also associated with neurological and respiratorysymptoms with a very low mortality rate Its clinical signsunder field conditions are those associated with drop inegg production and mild to moderate respiratory illness[48] Gross pathological findings are also minimal involvingonly a slight splenomegaly and other lesions as a resultof secondary bacterial infections Histopathological findingsinclude gliosis and perivascular cuffingwhichmay ormaynotbe accompanied by pancreatic necrosis [42]

The other forms of the disease are the lentogenic ND(LND) and asymptomatic enteric ND (AEND) which aregenerally associated with mild or no evidence of clinical dis-ease In fact the mild respiratory disease associated with theLND is only in young but not in adult chicken Experimentalinfection to study the pathology of lentogenic B1 and Q4strains in 4-week-old chicken produced no apparent clinicalsigns [60] Postmortem findings may be absent or at best mayinvolve mild hemorrhages in the tracheal and pulmonary


tissues At histology lymphoid follicles proliferation in thetracheal tissue might be encountered There may also be theloss of cilia infiltration of lymphocytes and squamous cellmetaplasia [61] Finally the AEND is completely avirulentcausing only the replication of the virus in the intestinaltissues of the infected chicken

322 Differential Diagnosis The clinicopathologic picture ofND gives important clues in making clinical diagnosis How-ever a number of viral and bacterial diseases may manifestsimilar clinical features that could be confused with NDThecommonest differentials of ND include highly pathogenicavian influenza infectious bronchitis infectious laryngotra-cheitis and diphtheritic formof fowl poxOthers include fowlcholera mycoplasmosis and psittacosis in psittacine avianspecies [62] Distinguishing ND from all these diseases is acrucial task in arriving at tentative diagnosis

33 Virus Isolation This is regarded as the gold standardmethod for the definitive diagnosis of ND and is oftenused for validating the results from other detection methods[63] The choice of samples required for virus isolation isdetermined by the sites of virus replication and routes of viralshedding In live birds samples required include the cloacaland oropharyngeal swabs collected in isotonic solution withor without antibiotics If the birds are already moribund orhave recently died samples should include lungs kidneyliver intestine spleen and caecal tonsils collected separatelyor as a pool in addition to the cloacal and oronasal swabs [2]To isolate NDV processed samples are primarily inoculatedinto the allantoic cavity of 9-10-day-old specific antibodyfree chicken embryonated eggs After about 4-7 days ofincubation hemagglutination test (HA) is used to detect thepresence of the virus in the infected allantoic fluid Howeversince other viruses such as avian influenza and APMVsmightalso possess HA activity it is always necessary to furtherconfirm the identity of the virus using other diagnostic testssuch as hemagglutination inhibition test (HI) using NDVspecific antisera or molecular tests It should be emphasisedthat some serological cross-reactivity might occur betweenNDV and APMV-3 or APMV-7 [63] This can however becircumvented by the use of a panel of monoclonal antibodiesspecific for NDV

Isolation of NDV can also be performed in primarycell cultures such as chicken embryo fibroblasts (CEF) DF-1 chicken embryo kidney (CEK) chicken embryo liver(CEL) cells and avian myeloblasts (QM5) which are allhighly permissive to the virus [64] The cells are infectedwith clinical samples and monitored for cytopathic effects(CPE) such as cell rounding syncytia formation and celldeath [65] Importantly isolation of avirulent NDV strainsin cell culture might require the addition of exogenoustrypsin since their monobasic F cleavage site can only beactivated by extracellular trypsin like proteases Worthy ofnote some pigeon adapted NDV strains (PPMV-1) can onlybe isolated via cell culture but not using embryonated eggs[6 7] Whenever such viruses are suspected it is advisableto attempt virus isolation in both embryonated eggs and cellculture Generally the use of cells in NDV isolation gives a

lower yield of the virus Hence even after virus isolation incells it might be necessary to propagate the isolated virus inembryonated eggs if the downstream application requires theuse of the virus in large quantities [32]

34 Serological Diagnosis The diagnostic relevance of serol-ogy in NDV surveillance is considerably belittled by itsinability to differentiate vaccinated from infected animals(DIVA) Nevertheless serological tests remain a valuable toolused by many diagnostic laboratories to assess the humoralimmune responses following vaccination [66] The simplestand most inexpensive serological test for NDV is HI whichmeasures the ability of NDV specific antibodies to inhibitthe agglutination of RBCs by the NDV particles The test isnormally performed using standard amount of NDV (4 or8 HAU) as HA antigen [67] The reciprocal of the highestserum dilution that completely blocks agglutination is the HItitre In birds whose HI titres are monitored closely (such asvaccinated birds) sudden rise in the titre might be indicativeof exposure to field NDV strain even though APMV-3 hasalso been reported to cause same [68]

Another robust test used in NDV serology is ELISAIn the last few years several ELISA kits based on wholeor part of the virus antigen have been developed for rapiddiagnosis of ND [69 70]Many of these kits are commerciallyavailable in the form of sandwich competitive or indirectELISA [44]They are highly sensitive and produce results thatpretty well correlate with HI test results Whereas in the HItests only the antibodies directed against the HN protein aredetected ELISA platforms utilising whole virus as antigenscan potentially detect antibodies directed against all theproteins in the NDV particle With the growing interests insubunit ND vaccines for disease control [71 72] it is possibleto develop ELISA tests that can differentiate antibodies dueto vaccination from those due to infection Makkay et al [69]developed antibody detection ELISA using recombinant NPprotein expressed in insect cells as antigen and demonstratedthe powerful DIVA property of the test Similarly Zhao etal [73] demonstrated that ELISA based on recombinant fulllength NP expressed in bacterial cells was able to detectNDV antibodies with high sensitivity in sera obtained fromvaccinated birds even though some levels of cross-reactivitywith antibodies raised against other APMVs were observedInterestingly the cross-reactivity was completely eliminatedwhen only the C terminal extension of the NP was used asa diagnostic antigen [73] Certain limitations however makethese tests less routinely used compared to the HAHI testsApart from being expensive and unsuitable in the field thesemonoclonal antibody (Mab) based ELISAsmay not be able todetect certain strains of NDV that might have somemutationin the single epitope against which the monoclonal antibodywas raised Nevertheless they still remain good diagnostictests for ND surveillance

Virus neutralisation test (VNT) is yet another powerfulserological test particularly useful inmeasuring NDV specificneutralising antibodies The test is performed by mixing aserially diluted serum with a standard amount of NDV (forinstance 100 PFU) followed by infection of the cultured DF-1 cells with the virus-serum mixtures [74] NDV specific


neutralising antibody titre is determined by the highestserum dilution that demonstrated a clear CPE in the culturedDF-1 cells after about four days of incubation The test isthe most superior tool for assessing neutralising antibodytitre following vaccination However it is highly laboriousand very slow yielding results only after nearly one weekInterestingly Chumbe et al [75] reported the developmentof an improved VNT using a recombinant NDV engineeredto constitutively express GFP The newly developed assay hasbeen shown to give conclusive resultswithin 24 hourswithoutthe need for any additional staining procedure Furthermoreits correlation with conventional VNT is much higher thanhow HI or ELISA is correlated with the conventional VNTThe test is therefore an emerging rapidmethod of quantifyingneutralising antibody titre However it will be better suited inthe assessment of vaccine protective immunity than in NDVsurveillance

35 Molecular Based Assays

351 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) The shortcomings of the conventional diagnostictechniques warrant the need for the development of morerapid yet very accurate methods of ND diagnosis in poultryThe most commonly used molecular test in NDV diagnosisespecially in the developing countries is RT-PCR The testcan rapidly and accurately detect viral genome in clinicalsampleswith high sensitivity especially if appropriate samplesare taken It is usually designed to simultaneously detect andidentify the pathotype of the virus [76] by targeting the Fgene portion encompassing the F cleavage site followed byrestriction fragment length polymorphism using BglI whosedigestion pattern classifies NDV isolates into lentogenicmesogenic and velogenic strains [77] Nowadays molecularNDV pathotyping is predominantly based on RT-PCR fol-lowed by the analysis of the putative amino acid compositionof the F cleavage site [78] Molecular based pathotypingis therefore a good alternative to the conventional virusisolation technique which in addition to being slow mightalso require specialised containment facilities [79] Howevergiven the continuous emergence and evolution of NDV [80]there is a need to regularly update the primers used in theassay so as to account for the variants that might escapedetection as a result of mutation in the primer binding site

352 Quantitative Polymerase Chain Reaction (qPCR) TheqPCR assay is not only faster and less cumbersome thanthe conventional diagnostic techniques but also providesequal or even greater sensitivity of virus detection than thegold standard virus isolation method In many countriesincluding the United States matrix gene and fusion genebased qPCR assays are often used as standard methodsfor NDV screening and pathotyping directly from clinicalsamples [81] The choice of matrix gene detection in NDVscreening is informed by its highly conserved nature in theNDV genome Thus matrix gene assay is able to detect mostof NDV isolates especially those that belong to class II [49]Because most of the class I isolates often escape detectionusing this assay an improved assay called matrix-polymerase

multiplex qPCR was developed by utilising a conservedregion on class I L gene for primer and probe design Thisnew assay not only detected the previously undetectableNDVisolates but also works in conjunction with the matrix geneassay under the same experimental settings [5] Matrix geneassay is therefore a rapid test for NDV screening in manycountries

For NDV pathotyping an F gene based qPCR assay thatdifferentiates the low virulence viruses from the virulentNDV strains was developed [82 83] Although this assay wasdesigned to detect NDV isolates in the United States it wasfound to be useful in detectingmost isolates around theworldexcept a few ones that possess nucleotide substitutions atthe probe binding sites [84] Further investigation revealedthat some of the isolates that escaped detection using thisprimer and probe combination had at their F cleavage site alysine (K) residue at position 114 instead of the conventionalglutamine (Q) at that position Indeed a number of othernucleotide differences were observed between the genome ofthose viruses and the F gene probe used in this assay Inter-estingly when a new probe that took the above nucleotidedifferences into consideration was designed and tested iso-lates that initially escaped detection using the earlier F geneassay were all identified with this improved platform [4]In our laboratory we recently developed a highly sensitiveprobe based qPCR system for the identification of virulentNDV strains circulating inMalaysia [18] Although the qPCRassay is currently validated for NDV screening from clinicalsamples it is imperative to continuously monitor the geneticdiversity of the evolving NDV isolates so that the primers andprobes can periodically be updated to identify all the possibleescape mutants Indeed a natural recombinant strain NDVIBS02513 isolated recently in our laboratory was shown toescape detection using our established primers and probesdue to a two-nucleotide substitution at the extreme 31015840 endof the probe binding site [85] When the probersquos sequencewas updated all the virulent NDV strains were successfullydetected

In addition to disease identification and pathotyping theqPCR assay can also be used in the quantification of viral loadin different organs [86] or virus shedding from the vaccinatedanimals following challenge with the virulent NDV strain [1887] The traditional methods of virus shedding assay are theend point dilutions such as mean tissue culture infective dose(TCID

50) and median egg infective dose (EID

50) or plaque

assay [88]Thesemethods are very laborious requiring a largenumber of eggs or multiple plates of seeded cells for themto be fully accomplished It also takes several days for theseassays to be completed Hence emerging trends in this regardinclude the use of qPCR systems which are highly sensitivespecific and reliable in the detection and quantification ofvirus in clinical samples within just a couple of hours

353 Non-PCR Amplification Techniques Recently a simplesensitive and inexpensive diagnostic assay called loop medi-ated isothermal amplification (LAMP) test was developedfor the rapid detection of the genetic materials of infectiousagents The principle of the assay is a strand displacementreaction that forms a stem loop structure allowing sensitive


and specific amplification of the target template [89] Thespecificity of LAMP test is due to its ability to detect sixindependent regions during the amplification reaction [9091] The assay is performed under isothermal conditions andonly requires 4-6 primers a DNA polymerase and a waterbath [92] It is therefore highly applicable to diagnostic labo-ratories in the developing countries wheremore sophisticatedinstruments are not readily available In addition the LAMPamplified product can easily be detected with naked eyes by asimple colour change To facilitate results visualisation SYBRgreen is traditionally added to the LAMP mixture prior tothe isothermal incubation [93] Pham et al [94] developeda LAMP based assay for the detection of NDV directly fromclinical samples and showed that its sensitivity and specificityare similar to those of nested PCR yet it is simpler andinexpensive Similarly Kirunda et al [95] reported the useof RT-LAMP to detect NDV RNA from cloacal and trachealswabs obtained from chicken in less than one hour Thusthe inexpensiveness rapidity specificity and sensitivity ofthe LAMP assay have made it highly useful in ND diagnosisespecially in the rural areas However the difficulties in thedesign of the four primers that independently target differentsites on the template as well as the challenges in LAMPmultiplexing are some of the limitations of this assay

36 Microarray Hybridisation Techniques Molecular baseddiagnostics have no doubt revolutionised the world of infec-tious disease diagnosis by simultaneously being simple rapidand sensitive in identifying disease agents However most ofthese assays may only detect a single or few organisms at atimeMicroarrays have the potential to concurrentlymonitordetect and characterise hundreds to thousands of targetswithout compromising the assayrsquos sensitivity and specificity[96]They are essentially made up of solid supports equippedwith several DNA probes of known identities capable ofhybridising to their targets in clinical samples Thus they aresaid to be efficient platforms for pathotyping genotyping anddisease biomarkers identification A lot of DNA microarraysystems have been developed for typing pathogenic agents[97 98] Using these microarray hybridisation systems Lunget al [99] reported the typing of NDV with a detectionlimit of as low as 101ndash103 TCID

50ml Indeed NDV and

avian influenza virus were simultaneously detected usingthis technique [99] demonstrating the potential of DNAmicroarrays in the detection of mixed infection DifferentNDV pathotypes as well as the H5 and H7 avian influenzaviruses (AIV)were also detected simultaneously using aDNAmicroarray hybridisation system developed by Wang et al[100] More recently newly developed microarray diagnos-tics including the multiplex luminex suspension microarraysystems were used for the simultaneous detection of NDVAIV infectious bursal disease virus and infectious bronchitisvirus in either single or mixed infections [101 102]Thereforeit will not be out of place to assert that DNA microarrays areamong the emerging diagnostics that in the near future couldoutperform the conventional NDV typing techniques giventheir sensitivity specificity and the ability to simultaneouslydetect multiple pathogenspathotype in clinical samples

37 Biosensor Diagnostics Biosensors are analytical systemsmade up of biorecognition molecules and physicochemicaldetectors called transducers capable of converting biomolec-ular interactions into measurable signals [103] They providean incredibly sensitive and inexpensive platform for the rapiddetection and identification of infectious disease agents [104]Label-free biosensors which provide ameans of continuouslymonitoring in real time the binding affinity and kineticsof biomolecules over time [105] are among the emergingdiagnostic tools in the 21st century Typical example is thesurface plasmon resonance (SPR) which directly quantifiesthe biomolecular interaction on its immobilised gold surfacevia the detection of changes in refractive index Using thisSPR technology a number of bacteria [106 107] viruses [108109] and other infectious agents [110] have been rapidly andselectively diagnosed Recently a label-free immunosensingsystem using excessively tilted fibre grating coated with goldnanospheres was developed The system is highly sensitivecapable of detecting as little as 5 pg of NDV [111] It is thusmore sensitive than the qPCR assay whose detection limit isaround 10 pg of the virus Furthermore this immunosensingmethod has a short detection time and does not requireany sophisticated equipment Therefore considering theiradvantages over other diagnostic technologies for NDVit is envisaged that the clinical application of biosensorswill improve in the nearest future So far translating thisbiosensor technology from the detection in laboratory solu-tions to direct clinical samples remains a major challengeperhaps due to the complex matrices that characterise theclinical samples [104] Notably most biosensors are highlysensitive to environmental factors such as temperature andpH Nevertheless several approaches are being developed toovercome those sample matrices effects

38 Next-Generation Sequencing Next-generation sequenc-ing (NGS) is one of the most recent tools that have revolu-tionised the diagnosis of infectious diseases [112] It is notonly important in tracking disease epidemics it also facilitiesthe rapid sensitive and specific detection and differentiationof mixed infections within a single host [113] It can also beused to detect low frequency variants which would otherwiseescape detection using the common diagnostic tools Inaddition it is currently the most throughput tool used inthe discovery of novel viruses associated with unknowndiseases [112] Hence its application in the development ofadvanced diagnostics cannot be over emphasised Althoughdifferent platforms of NGS are constantly emerging they alltechnically involve three major steps sample preparationsequencing and data analysis [114] Variations exist largely inthe sequencing techniques Currently most NGS platformsaimed at viral diagnosis are focusing on improved sequencereads and speed of the assay Recently NGS based character-isation of genotype VI NDV in the United States has beenreported to reveal a previously unknown genetic diversityof the virus with evidence of its continuous evolution [115]Satharasinghe et al [85] also discovered a naturally occurringhybrid NDV in Malaysia using NGS techniques with over99 coverage In addition NGS has recently been appliedto simultaneously characterise the genomic sequences of


multiple avian paramyxoviruses [116] More so differenti-ation of virulent from avirulent strains of class II NDVwas successfully and rapidly achieved using NGS based onpyrosequencing procedure [117] These features altogethermake NGS the most promising emerging technique for NDVdiscovery and diagnosis So far the only disadvantage of theassay is its perceived high cost of sample run However withthe increasingly cheaper sequencing services NGS tools arelikely to become routinely available for viral diagnosis giventheir speed accuracy and multiplexing ability

39 Random Priming Technologies Identification of newviruses using the conventional RT-PCR or even qPCR issolely dependent on the assumption that the unknown virusis somehow identical to the previously sequenced viruses atleast around the so-called conserved regions However thisis not always the case owing to the high genetic diversity andevolution of RNA viruses [14 118] Consequently periodicalmodification of the existing assays is necessary to account formutants that could escape detection using these molecularbased assays Furthermore in the case of mixed infectionthese assays will only amplify genomes that more closelymatch the primers and probes and not necessary the mostabundant in the pool of the samples [81] Therefore in orderto overcome these challenges it is necessary to develop morereliable and sensitive assays that are more robust than theexisting ones

Random priming techniques such as sequence indepen-dent single primer amplification (SISPA) have the poten-tial to overcome these shortcomings They work on theprinciple of random amplification of the genomic RNAsequencing assembly and analysis of the sequence reads[119] Using this SISPA method full genome coverage ofseveral viruses has been achieved within a short time andat a cost effective rate [120 121] However for effectiveresults it is always essential that the samples contain largenumber of virus particles In addition pretreatment of thesamples with DNAse-I may reduce the contamination fromthe host genome [119] Interestingly with the availability ofnext-generation sequencing (NGS) platforms that allow thedetection of low frequency variants in a pool of samples[122] SISPA technology can be greatly enhanced IndeedChrzastek et al [123] reported the use of SISPA in conjunctionwith NGS to identify NDV in a mixture of viruses whereits concentration is as low as 3 log 10 EID

50 As a matter of

fact complete genome sequence of NDV was obtained fromsamples containing mixed viruses where the titre of NDVwas only 45 log 10 EID

50[123] Thus rapid and accurate

identification of NDV can be achieved using this systemespecially under outbreak situation where thousands of birdsmight be at risk of dying due to the disease At presentthe use of this assay is limited to virus discovery and othermetagenomics applications because of its high cost per runHowever with the increased advancement in sequencingtechnologies the assay is expected to be much cheaper inthe nearest future and more available as a routine clinicaldiagnostic test

4 Vaccination Strategies

Due to their role in minimising the traffic of pathogenicorganisms in and out of the poultry facilities tight biosecuritybarriers are indispensable in the control of avian diseases[124] However even with a good biosecurity practicevaccination is still required to optimally protect the birdsagainst the economically devastating diseases such as NDHence effective poultry disease control program demands acombination of strict biosecurity measures with vaccinationregimen The strategies for ND vaccination are broadlyclassified into two the conventional methods developed inthe 1940s as well as the recently emerged methods based onrecombinant DNA technology Here a concise descriptionof these methods is provided with particular reference tostrengths and weaknesses of each method

41 Conventional Vaccines

411 Live Attenuated Vaccines The isolation of naturallyoccurring highly immunogenic NDV strains of low orintermediate virulence in the last 7 decades has certainlyameliorated the economic losses due toND inmany countries[125 126] To date a number of lentogenic NDV strainssuch as B1 F LaSota V4 and I

2are extensively used as

live vaccines for disease control [44] Among these vaccinestrains LaSota is obviously the most widely used in differentcountries because of its superior immunogenicity B1-basedlive ND vaccines may not be as immunogenic as LaSotabut are renowned for their highly attenuated feature with nopostvaccinal respiratory reactions in birds In the case of V4and I2 their major selling point is thermostability they can

tolerate elevated temperatures in the absence of cold chainmaking them especially valuable in villages with limitedrefrigeration capabilities [127] Other NDV strains used aspopular live vaccines include the Komorov and Mukteswarstrains both of which are mesogenic and therefore suitableas booster vaccines following primingwith lentogenic isolates[128]

All live attenuated ND vaccines are known to stimulateboth mucosal and systemic immune responses similar tothose of the natural infection because of their ability toreplicate in chicken irrespective of the site of administration[129] Furthermore a single administration of 105 EID

50

of live NDV vaccine is enough to rapidly stimulate animmune response capable of inducing a 100 protectionagainst clinical disease even though the shedding of thechallenged virulent virus via the cloacal and oropharyn-geal routes may still occur In fact there are indicationsthat the replication and shedding of the virulent virus canbe substantially reduced when much higher doses of thelive vaccines are administered [21 22] As this is not aneconomically viable option because of the high cost ofvaccination per bird improved and cost effective vaccinationapproaches are required to tackle the problem of virusshedding associated with the conventional ND vaccinesAn important factor that determines the effectiveness ofvaccination is the tissue tropism of the vaccine Conventionallive attenuated NDV vaccines including the famous LaSota


strains are predominantly respirotropic inducing strongermucosal immunity in the respiratory airways where initialexposure to the virus might occur Other vaccines such as theVGGA strains are more of enterotropic [130] stimulating gutmucosal immunity among the vaccinated birds Interestinglywe isolated a natural recombinant virulent NDV strainIBS02513 [85] which demonstrates a strong tissue tropismin both the respiratory and gastrointestinal systems It is ourbelief that this dual tropism feature of the virus and its highyield in chicken embryonated eggs make it a good candidatefor future development of live attenuated vaccines against NDin chickens

Perhaps the greatest advantage of live NDV vaccines istheir suitability for mass application via drinking water orspray making their total cost from the point of productionto administration highly inexpensive [131] In addition thevaccine virus from the vaccinated birds may spread to thesuboptimally vaccinated ones in the vicinity thereby con-tributing to the overall herd immunity [132] Furthermoresome of these attenuated strains such as NDV strain I

2have

been shown to be naturally thermostable [127] requiring littleor no cold chain maintenance making them suitable vaccinecandidates in the rural areas where electricity supply can begrossly inadequate Nevertheless in spite of all the benefitsof these vaccines they have their shortcomings First andforemost the live viruses may possess the potential to revertback to virulence and cause clinical disease in the vaccinatedbirds Secondly depending on the vaccine strain used thesevaccines may induce postvaccination respiratory reactions inyoung birds which if severe could predispose the birds tosecondary bacterial infections [133] In addition the vaccinesare largely based on genotypes I or II strains which are phylo-genetically divergent from the currently prevalent genotypesin different countries Hence although the vaccines are stillprotective against clinical signs and mortality caused byany NDV isolate their inability to block virus sheddingpostchallenge ensures the continuous presence of the virulentvirus in the environment This is particularly more dangerouswith genotype VII isolates whose shedding from LaSotavaccinated birds is significantly higher than those of othergenotypes [18] Therefore considering the above limitationsthe live attenuated vaccines must be used with utmost careand that the state-of-the-art vaccines are urgently needed toaddress these weaknesses of the conventional live attenuatedvaccines

412 Inactivated Vaccines Immunisation of chicken withinactivated vaccines is the earliest strategy for ND controlThe vaccines are produced by growing any NDV strain ofinterest to high titres followed by its inactivation using phys-ical or chemical methods [134] Since the viral surface gly-coproteins (F and HN) are the most important determinantsof neutralising antibodies methods used in viral inactivationshould be those that spare the immunogenic epitopes of thoseproteins Among the chemicals used for the inactivationbinary ethylenimine (BEI) and formaldehyde are the mostpopular [135] The vaccines are normally prepared in emul-sions of mineral oil (water-in-oil) and administered intra-muscularly or subcutaneously It is generally required that the

ratio of water phase to oil phase strikes a balance betweenthe vaccine stability and its viscosity such that the vaccineremains stable and yet not difficult to administer due to highviscosity of the emulsion [136] Because these vaccines cannotreplicate and spread horizontally among the vaccinated birdsthey are not suitable for mass application Rather they areadministered individually preferably via the parenteral routemaking the entire process hectic and expensive The samenonreplicating feature however makes them safe with no riskof reversion back to virulence [137]Therefore it is imperativeto optimise the conditions for developing these vaccinesas ldquotoo much inactivationrdquo may destroy the immunogenicepitopes and mild exposure to the chemical or irradiationmay not be sufficient to inactivate the virus [135] For bestresults these vaccines are administered after initial primingwith live vaccines and may require adjuvants to aid intailoring the immune responses to the immunodominantepitopes [137] Unfortunately these adjuvants may as wellcause some undesirable reactions in the vaccinated birdsAnother disadvantage of the inactivated vaccines is therequirement of a withdrawal period before birds immunisedwith those vaccines can be processed for human consumption[15] Additionally the inactivated ND vaccines are generallypoor inducers of mucosal or cell mediated immune response[137] Thus to ensure effective protection of chicken againstND rationally designed vaccines with improved margins ofsafety and efficacy are required in the poultry industry

42 Recombinant Vaccine Technologies

421 DNA Vaccines Advances in recombinant DNA tech-nology have made it possible to develop DNA vaccines bycloning a gene encoding an immunogen or group of neutral-ising epitopes into an expression plasmidWhen the recombi-nant plasmid is administered into the animal host the clonedgene can be transcribed and later translated into proteinwhich when processed by the host cells could serve as potentepitopes capable of inducing protective immune response[138] In a recent study where the complete NDV F gene wascloned in pIRES expression plasmid and used asDNAvaccinein chicken high antibody titre was observed In additionwhen a plasmid encoding both the F and HN proteins wasused as primer to vaccinate chicken and later boosted withinactivated NDV vaccine a superior protective antibodymediated immunity was observed [139] indicating that theseDNA vaccines can be used to improve the effectiveness ofinactivated NDVvaccinesThe effectiveness of DNAvaccinesmay be further enhanced when a vehicle such as nanoparticleis used to deliver the vaccine Firouzamandi et al [140] useddextran-spermine nanoparticle to encapsulate DNA vaccineencoding NDV F and HN proteins When the nanoencap-sulated vaccine was administered in ovo improvement inHI antibody titre was noticed although not significantlydifferent from the HI titre obtained from subjects vaccinatedwith the naked DNA vaccine Furthermore immunisationof SPF chicken with a chitosan encapsulated DNA vaccineencoding the NDV F gene leads to an enhanced mucosal andsystemic humoral and cell mediated responses [141] Thusthe DNA vaccine may represent a safe alternative vaccine


platform against the current ND challenges The majorselling points of these vaccines are their safety and ability toinduce both CD4+ and CD8+ immune responses Howevertheir limitations include poor immunogenicity high cost ofproduction and unsuitability for mass administration Moreso when administered without any delivery vehicle they areeasily degraded by the nucleases before they reach their finaldestination Nonetheless the use of adjuvants and deliveryvehicle may overcome some of these limitations [142 143]

422 Viral Vector Vaccines One of the most promisingstrategies used to combat veterinary andmedically importantpathogens is the use of recombinant viral vector vaccinesThemost common vectors used in poultry are the vaccinia virusfowl pox virus and herpes virus of turkeys [144 145] Becauseof their large double stranded DNA genome vaccinia viruseshave a very high capacity of foreign gene expression Theyare highly immunogenic capable of inducing strong inflam-matory innate immune response via the TLR activation Fur-thermore they can be easily propagated in large scale usingchicken embryo fibroblast cells Hence they are frequentlyused as gene delivery tools against cancer and so many otherinfectious diseases Since early 1990s recombinant vacciniavirus expressing the F gene from NDV strain Italian wasshown to induce strong immunity that protected birds againstvirulent NDV challenge [146] However the limitation of thisvector such as sensitivity to preexisting immunity against thevector [147] makes its usage in ND vaccine delivery highlylimited

Recombinant fowl pox vectored ND vaccines generatedby replacing the thymidine kinase gene with NDV F HN orF and HNwere shown to induce protective immunity againstthe virulent NDVchallenge in chicken [148 149]The greatestadvantage of the fowl pox vectored vaccine is its inabilityto cause postvaccinal respiratory reactions in the vaccinatedchicken However presence of anti-fowl pox virus antibodiesin the vaccinated chicken may dramatically interfere withthe efficiency of this vector Furthermore this vaccine maynot be suitable for young birds Interestingly this particularshortcoming can be overcome by the use of recombinantherpes virus vectored vaccine which can directly be used inboth 18-day-old embryo and 1-day-old chicks [150] It elicits astrong and long lasting cell mediated and humoral immunitybecause of its ability to persist as a latent infection in thevaccinated chicken Furthermore its gene delivery efficiencyis not adversely affected by the presence of preexistingimmunity against the backbone vector because it replicatesin a kind of cell associated manner [151] These propertiescollectively make the vector an ideal vehicle for the deliveryNDV immunogens Indeed recombinant HVT expressing theNDVFglycoproteinwas shown to induce 95-100protectionof chicken about 4 weeks after vaccination via the in ovo orsubcutaneous routes [152] Thus vaccination against virulentND using the HVT vectors remains a promising system foreffective disease control in poultry

Other viral vectors used to deliver NDV vaccines includethe avian paramyxovirus-3 (APMV-3) This virus has beenshown to replicate excellently in chickens turkeys and evencell culture [153] The virus has also been shown to be highly

attenuated in chicken with ICPI value less than those ofmost lentogenic NDV isolates [153] Importantly preexistingimmunity against NDV does not interfere with its efficiencyas a vaccine vector Thus APMV-3 is an alternative viralvector that can efficiently infect chicken without causingany clinical disease Recently Kumar et al [154] constructedrecombinant APMV-3 vaccines vectoring either NDV F orHN When these vaccines were used to vaccinate 2-week-old chicken NDV specific cellular and humoral immuneresponses which protected against virulent NDV challengewere observed It is however worth mentioning that express-ing NDV F or HN in APMV-3 backbone lead to a retardationof replication of the chimeric viruses compared to the wildtype APMV-3 Nevertheless APMV-3 is still regarded as anefficient avirulent vaccine vector in chicken

423 Virus Like Particles Platforms Virus like particles(VLPs) are genome less assemblies of virus structural pro-teins made up of repetitive surface structures that serve aspathogen associated molecular patterns capable of elicitinga robust immune response [155] They are morphologicallyvery similar to viruses but are replication incompetentmaking them a highly safe vaccine platform [156] A fewyears ago production of NDVLPs was achieved following theexpression of M protein in combination with the NP andviral surface glycoproteins (F and HN) [157] More so coex-pression of NDV F protein and avian influenza M1 proteineffectively produced VLPs in baculovirus expression system[158] Interestingly in the above studies not only did theVLPssuccessfully incorporate the surface glycoproteins but theproteinsrsquo structural conformation and biological functionssuch as fusogenicity hemagglutination and neuraminidaseactivities remained unaffected Additionally immunisationof mice or chicken with the VLPs induced strong immuneresponses similar to those of equivalent amount of inactivatedND vaccines [158 159] There are several unique featuresthat differentiate NDVLPs from many other VLP systemsFirst and foremost the ratio of the proteins in the VLPsis very similar to that in the wild type virus Secondlyunlike other VLPs that are released with efficiencies of 10-50 the NDVLPs were shown to be released with efficiencyof 84 from the avian cells [157] making them the VLPswith the highest known release efficiency Furthermore theNDVLPs can easily be concentrated and purified to be devoidof any cell content contamination using the establishedprotocols for virus purification Unfortunately producingvery large amount of VLPs for massive vaccine trial mightbe challenging especially if platforms other than baculovirusexpression systems are used Furthermore since VLPs cannotreplicate in the vaccinated hosts they need to be administeredindividually in large quantities and with adjuvants in order toachieve effective immune response against the disease [160]Despite all these challenges VLPs are still promising safevaccine platforms that increasingly gain popularity in thecontrol of NDV

424 NDV Reverse Genetics-Based Vaccines The greatestweakness of the conventional genotype II based NDVvaccines is their inability to stop the shedding of the


heterologous virulent NDV even if clinical protection isachieved Although a lot of factors might be involved in thispostvaccination shedding of virulent NDV genotype mis-match between the vaccine and challenge strains is believedto be a key player Experimentally it has been shown thatvirus shedding can substantially be reduced when birdsare immunised with vaccines which are homologous to thechallenge strainsHence the newdirection in the fight againstND focuses on the generation of the so-called genotype-matched NDV vaccines

The latest strategy for the development of genotype-matched live attenuated ND vaccines is reverse geneticswhich is the recovery of a recombinant virus from its clonedcDNA [161] Since the major virulence determinant of NDVhas been shown to be the F protein cleavage site [54] whoseamino acid composition clearly distinguishes virulent (poly-basic) from avirulent (monobasic) strains reverse geneticscan be used to generate genotype-matched ND vaccine bymodifying the cleavage site of the prevalent virulent NDVfrompolybasic tomonobasic [162] Using this approach Xiaoet al [163] genetically modified the F cleavage site of a highlyvirulent NDV circulating in Indonesia and showed that itcompletely lost its virulence and induces a superb protectiveimmunity that significantly reduced virus shedding followingchallenge with a highly virulent wild type genotype VII NDVisolate

In another study a highly virulent NDV strain JS5 wasused as a back bone to develop a genotype-matched vaccineagainst genotype VII NDV By changing the F cleavagesite of the virus from polybasic to monobasic the rescuedvirus completely lost its virulent phenotype but retainedits tropism in chicken embryonated eggs and induced aprotective immunity that lead to significant reduction in theshedding of the challenge virus compared to the conven-tional LaSota vaccine [88] Therefore reverse genetics is anattractive technology for rapid generation of stably attenuatedgenotype-matched vaccines against virulent NDV Anotherimportant application of this technology is in the generationofmarker NDV vaccines able to differentiate vaccinated fromthe infected animals (DIVA) These DIVA vaccines representan invaluable strategy for sustainable eradication of ND inpoultry [162] At present reverse genetics-based vaccinesare too expensive to develop because of the high cost ofsequencing and other molecular biology services Howeverwith the increasingly available gene synthesis industries thecost of these vaccines is likely to drastically reduce in thenear future Therefore the proliferation of these vaccines indifferent countries is strongly anticipated in the near futuregiven their unique characteristics such as protective efficacygenetic stability and homogeneity with the prevalent NDVstrains

5 Concluding Remarks

To effectively control virulent NDV infection in poultry it isimperative to rapidly and specifically identify the aetiologicagent Currently traditional virus isolation followed by itsserological or molecular confirmation is considered to be thegold standard method for NDV detection and pathotyping

[2] However the lengthy protocol of this assay and itsrequirement for specialised containment facility diminishesits diagnostic relevance especially under outbreak situationswhere prompt diagnosis and immediate intervention arerequiredMolecular based assays such as RT-PCR qPCR andLAMP can rapidly detect and differentiate NDV pathotypesbased on the chemistry of their F cleavage sites Unfor-tunately NDV virulence is multigenic and other factorsindependent of the F cleavage site might be important indetermining the actual virulence potential of the virus Thusthe biggest question in the midst of this diagnostic dilemmais lsquowhat is the best tool for rapid and accurate diagnosis ofvirulent NDV infectionrsquo Apparently the answer to this ques-tion is NGS which can specifically and rapidly identify allthe virulencemarkers in the viral genome and simultaneouslydifferentiate mixed infections within a single clinical sampleNGS is therefore themost promising emerging technique forthe diagnosis and pathotyping of virulent NDV isolates

An excellent NDVvaccine is that which not only preventsclinical disease but also reduces or abolishes virus sheddingand increases the quantity of the virulent virus required tocause infection [15] Unfortunately the currently availableinactivated and live attenuated NDV vaccines can onlyprevent clinical disease but not virus shedding especiallyfollowing heterologous virus challenge [18] Yet they remainin the mainstay of ND control for more than six decades dueto their ldquodisease preventing abilityrdquo and relatively cheap costof production Nevertheless the search for better alternativesstill continues and has so far lead to the emergence of novelvaccine platforms based on recombinant DNA technologyAmong these emerging vaccines theVLPs andDNAvaccinesare known for their incredible safety but sadly they are poorlyimmunogenic Recombinant viral vectored NDV vaccineshave also demonstrated promising protective efficacy but aresignificantly affected by the presence of maternally derivedantibodies against the vector So far the most promisingvaccines against the virulent NDV infection in poultry arethe recombinant genotype-matched live attenuated vaccinecandidates generated by reverse genetics They specificallytarget the prevailing genotype in a particular region andare therefore rationally designed to fulfil the criteria of anexcellent NDVvaccine It is envisaged that these vaccines willin the near future outshine all the currently available NDVvaccines

Conflicts of Interest

The authors declare that the work presented was not per-formed in the presence of any personal professional orfinancial relationships that could potentially be seen asconflicts of interest

Acknowledgments

Special appreciation goes to Dr Tan Sheau Wei and otherstaff of the Laboratory of Vaccines and ImmunotherapeuticsInstitute of Bioscience Universiti Putra Malaysia for theirinvaluable support and cooperation


References

[1] D J Alexander ldquoNewcastle diseaserdquo British Poultry Science vol42 no 1 pp 5ndash22 2001

[2] OIE ldquoNewcastle Disease (Infection with Newcastle DiseaseVirus)rdquo Manual of Diagnostic Tests and Vaccines for TerrestrialAnimals (Mammals Birds and Bees) vol 1 pp 555ndash574 2012

[3] K Murulitharan K Yusoff A R Omar and A MoloukildquoCharacterization of Malaysian velogenic NDV strain AF2240-I genomic sequence A comparative studyrdquo Virus Genes vol 46no 3 pp 431ndash440 2013

[4] L M Kim D J King H Guzman et al ldquoBiological and phy-logenetic characterization of pigeon paramyxovirus serotype 1circulating in wild North American pigeons and dovesrdquo Journalof Clinical Microbiology vol 46 no 10 pp 3303ndash3310 2008

[5] L M Kim D L Suarez and C L Afonso ldquoDetection of abroad range of class I and II Newcastle disease viruses usingmultiplex real-time reverse transcription polymerase chainreaction assayrdquo Journal of Veterinary Diagnostic Investigationvol 20 no 4 pp 414ndash425 2008

[6] J C F M Dortmans P J M Rottier G Koch and B PH Peeters ldquoPassaging of a newcastle disease virus pigeonvariant in chickens results in selection of viruseswithmutationsin the polymerase complex enhancing virus replication andvirulencerdquo Journal of General Virology vol 92 no 2 pp 336ndash345 2011

[7] J C Dortmans G Koch P J Rottier and B P PeetersldquoVirulence of newcastle disease virus what is known so farrdquoVeterinary Research vol 42 no 1 article no 112 2011

[8] J C F M Dortmans P J M Rottier G Koch and B P HPeeters ldquoThe viral replication complex is associated with thevirulence of newcastle disease virusrdquo Journal of Virology vol84 no 19 pp 10113ndash10120 2010

[9] J Jin J Zhao Y Ren Q Zhong and G Zhang ldquoContributionof HN protein length diversity to Newcastle disease virus vir-ulence replication and biological activitiesrdquo Scientific Reportsvol 6 2016

[10] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[11] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985

[12] K Tamura M Nei and S Kumar ldquoProspects for inferringvery large phylogenies by using the neighbor-joining methodrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 101 no 30 pp 11030ndash11035 2004

[13] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 Molecular Evolutionary Genetics Analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013

[14] C J Snoeck A A Owoade E Couacy-Hymann et al ldquoHighgenetic diversity of newcastle disease virus in poultry inwest and central Africa Cocirculation of genotype XIV andnewly defined genotypes XVII and XVIIIrdquo Journal of ClinicalMicrobiology vol 51 no 7 pp 2250ndash2260 2013

[15] D R Kapczynski C L Afonso and P J Miller ldquoImmuneresponses of poultry to Newcastle disease virusrdquoDevelopmentalamp Comparative Immunology vol 41 no 3 pp 447ndash453 2013

[16] D PerelmanW F Goldman and G Borkow ldquoEnhancement ofantibody titers against Newcastle Disease Virus in vaccinatedchicks by administration of Phyto V7rdquo Journal of Vaccines andVaccination vol 4 no 7 2013

[17] JM Sharma ldquoIntroduction to poultry vaccines and immunityrdquoAdvances in Veterinary Medicine vol 41 no C pp 481ndash4941999

[18] K Roohani S W Tan S K Yeap A Ideris M H Bejo and AR Omar ldquoCharacterisation of genotype VII Newcastle diseasevirus (NDV) isolated from NDV vaccinated chickens andthe efficacy of LaSota and recombinant genotype VII vaccinesagainst challenge with velogenic NDVrdquo Journal of VeterinaryScience vol 16 no 4 pp 447ndash457 2015

[19] S F Rehmani A Wajid T Bibi et al ldquoPresence of virulentnewcastle disease virus in vaccinated chickens in farms inPakistanrdquo Journal of Clinical Microbiology vol 53 no 5 pp1715ndash1718 2015

[20] L M Thomazelli J De Araujo T Fabrizio et al ldquoNovel avianparamyxovirus (APMV-15) isolated from a migratory bird inSouth Americardquo PLoS ONE vol 12 no 5 Article ID e01772142017

[21] K M Dimitrov C L Afonso Q Yu and P J Miller ldquoNew-castle disease vaccinesmdashA solved problem or a continuouschallengerdquoVeterinary Microbiology vol 206 pp 126ndash136 2016

[22] K M Dimitrov A M Ramey X Qiu J Bahl and C LAfonso ldquoTemporal geographic and host distribution of avianparamyxovirus 1 (Newcastle disease virus)rdquo Infection Geneticsand Evolution vol 39 pp 22ndash34 2016

[23] A Czegledi D Ujvari E Somogyi E Wehmann O Wernerand B Lomniczi ldquoThird genome size category of avianparamyxovirus serotype 1 (Newcastle disease virus) and evolu-tionary implicationsrdquoVirus Research vol 120 no 1-2 pp 36ndash482006

[24] M Steward I B Vipond N S Millar and P T EmmersonldquoRNA editing in Newcastle disease virusrdquo Journal of GeneralVirology vol 74 no 12 pp 2539ndash2547 1993

[25] K Yusoff and W S Tan ldquoNewcastle disease virus macro-molecules and opportunitiesrdquo Avian Pathology vol 30 no 5pp 439ndash455 2001

[26] A J Battisti G Meng D C Winkler et al ldquoStructure andassembly of a paramyxovirus matrix proteinrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 109 no 35 pp 13996ndash14000 2012

[27] K K Conzelmann ldquoReverse genetics of MononegaviralesrdquoCurrent Topics in Microbiology and Immunology vol 283 pp1ndash41 2004

[28] R P Hanson and C A Brandly ldquoNewcastle Diseaserdquo Annals ofthe New York Academy of Sciences vol 70 no 3 pp 585ndash5971958

[29] D J Alexander E W Aldous and C M Fuller ldquoThe long viewa selective review of 40 years of Newcastle disease researchrdquoAvian Pathology vol 41 no 4 pp 329ndash335 2012

[30] G L Pearson andM K McCann ldquoThe role of indigenous wildsemidomestic and exotic birds in the epizootiology of velogenicviscerotropic Newcastle disease in Southern California 1972-1973rdquo Journal of the American Veterinary Medical Associationvol 167 no 7 pp 610ndash614 1975

[31] E E Grass ldquoExotic Newcastle disease in the United Statesrdquoin Proceedings of the 21st Western Poultry Disease Conferenceand 6th Poultry Health Symposium pp 26ndash30 University ofCalifornia Davis CA USA 1972

[32] J T Lumeij and J W Stam ldquoParamyxovirus disease in racingpigeons Clinical aspects and immunization A report from theNetherlandsrdquoVeterinaryQuarterly vol 7 no 1 pp 60ndash65 1985


[33] S W Tan A Ideris A R Omar K Yusoff and M Hair-Bejo ldquoSequence and phylogenetic analysis of Newcastle diseasevirus genotypes isolated in Malaysia between 2004 and 2005rdquoArchives of Virology vol 155 no 1 pp 63ndash70 2010

[34] J Herczeg E Wehmann R R Bragg et al ldquoTwo novel geneticgroups (VIIb andVIII) responsible for recentNewcastle diseaseoutbreaks in Southern Africa one (VIIb) of which reachedSouthern EuroperdquoArchives of Virology vol 144 no 11 pp 2087ndash2099 1999

[35] H-J Kwon S-H Cho Y-J Ahn S-H Seo K-S Choi and S-JKim ldquoMolecular epidemiology ofNewcastle disease in Republicof Koreardquo Veterinary Microbiology vol 95 no 1-2 pp 39ndash482003

[36] P J Miller R Haddas L Simanov et al ldquoIdentification ofnew sub-genotypes of virulent Newcastle disease virus withpotential panzootic featuresrdquo Infection Genetics and Evolutionvol 29 pp 216ndash229 2015

[37] A J Ayala K M Dimitrov C R Becker et al ldquoPresence ofvaccine-derived newcastle disease viruses in wild birdsrdquo PLoSONE vol 11 no 9 Article ID e0162484 2016

[38] V R Brown and S N Bevins ldquoA review of virulent Newcastledisease viruses in the United States and the role of wild birds inviral persistence and spreadrdquoVeterinary Research vol 48 no 12017

[39] C Meng X Qiu S Yu et al ldquoEvolution of newcastle diseasevirus quasispecies diversity and enhanced virulence after pas-sage through chicken air sacsrdquo Journal of Virology vol 90 no4 pp 2052ndash2063 2016

[40] A Petrini and B Vallat ldquoNotification of avian influenza andnewcastle disease to the world organisation for animal health(OIE)rdquo Avian Influenza and Newcastle Disease A Field andLaboratory Manual pp 27ndash30 2009

[41] S Samal S Kumar S K Khattar and S K Samal ldquoA singleamino acid change Q114R in the cleavage-site sequence ofNewcastle disease virus fusion protein attenuates viral replica-tion and pathogenicityrdquo Journal of General Virology vol 92 no10 pp 2333ndash2338 2011

[42] C Brown D J King and B S Seal ldquoPathogenesis of Newcastledisease in chickens experimentally infected with viruses ofdifferent virulencerdquoVeterinary Pathology vol 36 no 2 pp 125ndash132 1999

[43] M Munir M Cortey M Abbas Z U A Qureshi F Afzal MZ Shabbir et al ldquoBiological characterization and phylogeneticanalysis of a novel genetic group of Newcastle disease virusisolated from outbreaks in commercial poultry and frombackyard poultry flocks in Pakistanrdquo Infection Genetics andEvolutions vol 12 no 5 pp 1010ndash1019 2012

[44] OIE ldquoNewcastle diseaserdquo Manual of Diagnostic Tests andVaccines for Terrestrial Animals (Mammals Birds and Bees) vol1 pp 576ndash589 2008

[45] M Farooq U Saliha M Munir and Q M Khan ldquoBiologicaland genotypic characterization of the Newcastle disease virusisolated from disease outbreaks in commercial poultry farms innorthern Punjab Pakistanrdquo Virology Reports vol 3 pp 30ndash392014

[46] D J Alexander and G Parsons ldquoPathogenicity for chickens ofavian paramyxovirus type i isolates obtained from pigeons ingreat britain during 1983ndash85rdquoAvian Pathology vol 15 no 3 pp487ndash493 1986

[47] A M Piacenti D J King B S Seal J Zhang and C C BrownldquoPathogenesis of newcastle disease in commercial and specific

pathogen-free Turkeys experimentally infected with isolates ofdifferent virulencerdquoVeterinary Pathology vol 43 no 2 pp 168ndash178 2006

[48] D J Alexander and D A Senne ldquoNewcastle disease virusand other avian paramyxovirusesrdquo in A Laboratory Manualfor the Isolation Identification and Characterization of AvianPathogens Dufour-Zavala D E Swayne and J R GlissonEds pp 135ndash141 American Association of Avian PathologistsJacksonville Fla USA 5th edition 2008

[49] L M Kim D J King P E Curry et al ldquoPhylogenetic diversityamong low-virulence newcastle disease viruses from waterfowland shorebirds and comparison of genotype distributions tothose of poultry-origin isolatesrdquo Journal of Virology vol 81 no22 pp 12641ndash12653 2007

[50] Y Kang Y Li R Yuan et al ldquoPhylogenetic relationshipsand pathogenicity variation of two Newcastle disease virusesisolated from domestic ducks in Southern Chinardquo VirologyJournal vol 11 no 1 article no 147 2014

[51] L Susta D Segovia T L Olivier et al ldquoNewcastleDiseaseVirusInfection in Quailrdquo Veterinary Pathology 2018

[52] J E Pearson D A Senne D J Alexander W D Taylor LA Peterson and P H Russell ldquoCharacterization of Newcastledisease virus (avian paramyxovirus-1) isolated from pigeonsrdquoAvian Diseases vol 31 no 1 pp 105ndash111 1987

[53] G Cattoli L Susta C Terregino and C Brown ldquoNewcastledisease a review of field recognition and current methods oflaboratory detectionrdquo Journal of Veterinary Diagnostic Investi-gation vol 23 no 4 pp 637ndash656 2011

[54] A Panda Z Huang S Elankumaran D D Rockemann and SK Samal ldquoRole of fusion protein cleavage site in the virulenceof Newcastle disease virusrdquoMicrobial Pathogenesis vol 36 no1 pp 1ndash10 2004

[55] O S de Leeuw L Hartog G Koch and B P H PeetersldquoEffect of fusion protein cleavage site mutations on virulenceof Newcastle disease virus Non-virulent cleavage site mutantsrevert to virulence after one passage in chicken brainrdquo Journalof General Virology vol 84 no 2 pp 475ndash484 2003

[56] M S Collins I Strong and D J Alexander ldquoEvaluation ofthe molecular basis of pathogenicity of the variant Newcastledisease viruses termed ldquopigeon PMV-1 virusesrdquordquo Archives ofVirology vol 134 no 3-4 pp 403ndash411 1994

[57] F S Marks C R Rodenbusch C H Okino et al ldquoTargetedsurvey of Newcastle disease virus in backyard poultry flockslocated in wintering site for migratory birds from SouthernBrazilrdquo Preventive Veterinary Medicine vol 116 no 1-2 pp 197ndash202 2014

[58] MD Falcon ldquoExotic Newcastle diseaserdquo Seminars in Avian andExotic Pet Medicine vol 13 no 2 pp 79ndash85 2004

[59] M Banerjee W M Reed S D Fitzgerald and B PanigrahyldquoNeurotropic velogenic Newcastle disease in cormorants inMichigan Pathology and virus characterizationrdquo Avian Dis-eases vol 38 no 4 pp 873ndash878 1994

[60] H Hamid R S F Campbell and C Lamichhane ldquoThePathology of Infection of Chickens With the Lentogenic V4Strain of Newcastle Disease Virusrdquo Avian Pathology vol 19 no4 pp 687ndash696 1990

[61] P T Hooper E Hansson J G Young G M Russell and A JDella-Porta ldquoLesions in the upper respiratory tract in chickensexperimentally infected with Newcastle disease viruses isolatedin AustraliardquoAustralian Veterinary Journal vol 77 no 1 pp 50-51 1999


[62] D J Alexander ldquoNewcastle disease diagnosisrdquo inNewcastle Dis-ease D J Alexander Ed vol 8 of Developments in VeterinaryVirology pp 147ndash160 Springer US Boston MA USA 1988

[63] D J Alexander ldquoNewcastle disease and other avian paramyx-ovirusesrdquo International Office of Epizootics vol 19 no 2 pp443ndash462 2000

[64] L W McGinnes H Pantua J Reitter and T G MorrisonldquoNewcastle disease virus propagation quantification and stor-agerdquo Current Protocols in Microbiology vol 15 pp 15F21ndash15F218 2006

[65] P V Ravindra A K Tiwari B Ratta U Chaturvedi S KPalia and R S Chauhan ldquoNewcastle disease virus-inducedcytopathic effect in infected cells is caused by apoptosisrdquo VirusResearch vol 141 no 1 pp 13ndash20 2009

[66] K-S Choi S-J Kye W-J Jeon et al ldquoPreparation and diagnos-tic utility of a hemagglutination inhibition test antigen derivedfrom the baculovirus-expressed hemagglutinin-neuraminidaseprotein gene of Newcastle disease virusrdquo Journal of VeterinaryScience vol 14 no 3 pp 291ndash297 2013

[67] G Cross ldquoHemagglutination inhibition assaysrdquo Seminars inAvian and Exotic Pet Medicine vol 11 no 1 pp 15ndash18 2002

[68] R Tsunekuni H Hikono and T Saito ldquoEvaluation of avianparamyxovirus serotypes 2 to 10 as vaccine vectors in chickenspreviously immunized against Newcastle disease virusrdquo Veteri-nary Immunology and Immunopathology vol 160 no 3-4 pp184ndash191 2014

[69] A M Makkay P J Krell and E Nagy ldquoAntibody detection-based differential ELISA for NDV-infected or vaccinated chick-ens versus NDV HN-subunit vaccinated chickensrdquo VeterinaryMicrobiology vol 66 no 3 pp 209ndash222 1999

[70] A Berinstein C Vazquez-Rovere S Asurmendi et al ldquoMucosaland systemic immunization elicited by Newcastle disease virus(NDV) transgenic plants as antigensrdquo Vaccine vol 23 no 48-49 pp 5583ndash5589 2005

[71] J Ge Y Liu L Jin D Gao C Bai and W Ping ldquoConstructionof recombinant baculovirus vaccines for Newcastle DiseaseVirus and an assessment of their immunogenicityrdquo Journal ofBiotechnology vol 10 pp 16ndash24 2016

[72] Y-J Lee H-W Sung J-G Choi et al ldquoProtection of chick-ens from Newcastle disease with a recombinant baculovirussubunit vaccine expressing the fusion and hemagglutinin-neuraminidase proteinsrdquo Journal of Veterinary Science vol 9no 3 pp 301ndash308 2008

[73] N Zhao C Grund M Beer and T C Harder ldquoEngineeredrecombinant protein products of the avian paramyxovirustype-1 nucleocapsid and phosphoprotein genes for serologicaldiagnosisrdquo Virology Journal vol 15 no 1 2018

[74] G KochG Czifra andB E Engstrom ldquoDetection ofNewcastledisease virus-specific antibodies in ostrich sera by three sero-logical methodsrdquo Veterinary Record vol 143 no 1 pp 10ndash121998

[75] A Chumbe R Izquierdo-Lara K Calderon M Fernandez-Dıaz and V N Vakharia ldquoDevelopment of a novel Newcastledisease virus (NDV) neutralization test based on recombinantNDV expressing enhanced green fluorescent proteinrdquo VirologyJournal vol 14 no 1 2017

[76] Z Wang F T Vreede J O Mitchell and G J ViljoenldquoRapid detection and differentiation of Newcastle disease virusisolates by a triple one-step RT-PCRrdquo Onderstepoort Journal ofVeterinary Research vol 68 no 2 pp 131ndash134 2001

[77] T Nanthakumar R S Kataria A K Tiwari G Butchaiah andJ M Kataria ldquoPathotyping of Newcastle Disease Viruses by

RT-PCRand Restriction Enzyme AnalysisrdquoVeterinary ResearchCommunications vol 24 no 4 pp 275ndash286 2000

[78] O S de Leeuw G Koch L Hartog N Ravenshorst and B PH Peeters ldquoVirulence of Newcastle disease virus is determinedby the cleavage site of the fusion protein and by both the stemregion and globular head of the haemagglutinin-neuraminidaseproteinrdquo Journal of General Virology vol 86 no 6 pp 1759ndash1769 2005

[79] A Haryanto M Purwaningrum S Verawati S H Irianingsihand N Wijayanti ldquoPathotyping of Local Isolates NewcastleDiseaseVirus fromField Specimens byRT-PCRandRestrictionEndonuclease Analysisrdquo Procedia Chemistry vol 14 pp 85ndash902015

[80] D G Diel L H A da Silva H Liu Z Wang P J Miller andC L Afonso ldquoGenetic diversity of avian paramyxovirus type 1Proposal for a unified nomenclature and classification systemof Newcastle disease virus genotypesrdquo Infection Genetics andEvolution vol 12 no 8 pp 1770ndash1779 2012

[81] P JMiller E L Decanini and C L Afonso ldquoNewcastle diseaseevolution of genotypes and the related diagnostic challengesrdquoInfection Genetics and Evolution vol 10 no 1 pp 26ndash35 2010

[82] EW AldousM S Collins AMcGoldrick andD J AlexanderldquoRapid pathotyping of Newcastle disease virus (NDV) usingfluorogenic probes in a PCRassayrdquoVeterinaryMicrobiology vol80 no 3 pp 201ndash212 2001

[83] T Farkas E Szekely S Belak and I Kiss ldquoReal-time PCR-basedpathotyping of newcastle disease virus by use of TaqManminorgroove binder probesrdquo Journal of Clinical Microbiology vol 47no 7 pp 2114ndash2123 2009

[84] C Terregino G Cattoli B Grossele E Bertoli E Tisato andI Capua ldquoCharacterization of Newcastle disease virus isolatesobtained from Eurasian collared doves (Streptopelia decaocto)in Italyrdquo Avian Pathology vol 32 no 1 pp 63ndash68 2003

[85] D A Satharasinghe K Murulitharan S W Tan et al ldquoDetec-tion of inter-lineage natural recombination in avian paramyx-ovirus serotype 1 using simplified deep sequencing platformrdquoFrontiers in Microbiology vol 7 pp 1ndash14 2016

[86] H G M Niesters ldquoQuantitation of viral load using real-timeamplification techniquesrdquoMethods vol 25 no 4 pp 419ndash4292001

[87] M Rasoli S K Yeap S W Tan et al ldquoAlteration in lymphocyteresponses cytokine and chemokine profiles in chickens infectedwith genotype VII and VIII velogenic Newcastle disease virusrdquoComparative Immunology Microbiology amp Infectious Diseasesvol 37 no 1 pp 11ndash21 2014

[88] Z Hu S Hu CMeng XWang J Zhu and X Liu ldquoGenerationof a genotypeVIInewcastle disease virus vaccine candidatewithhigh yield in embryonated chicken eggsrdquoAvianDiseases vol 55no 3 pp 391ndash397 2011

[89] T Notomi H Okayama H Masubuchi et al ldquoLoop-mediatedisothermal amplification of DNArdquo Nucleic Acids Research vol28 no 12 article no E63 2000

[90] K Nagamine T Hase and T Notomi ldquoAccelerated reactionby loop-mediated isothermal amplification using loop primersrdquoMolecular and Cellular Probes vol 16 no 3 pp 223ndash229 2002

[91] Z K Njiru ldquoLoop-mediated isothermal amplification technol-ogy Towardspoint of care diagnosticsrdquoPLOSNeglected TropicalDiseases vol 6 no 6 Article ID e1572 2012

[92] Y Mori and T Notomi ldquoLoop-mediated isothermal amplifi-cation (LAMP) A rapid accurate and cost-effective diagnos-tic method for infectious diseasesrdquo Journal of Infection andChemotherapy vol 15 no 2 pp 62ndash69 2009


[93] A Izadi E Moslemi and M H Shahhosseiny ldquoComparisonof SYBR Green and turbidimetry methods for loop mediatedisothermal amplification (LAMP) product detection in diagno-sis of hepatitis B virus (HBV)rdquo African Journal of MicrobiologyResearch vol 6 no 42 pp 7003ndash7007 2012

[94] H M Pham C Nakajima K Ohashi and M Onuma ldquoLoop-Mediated Isothermal Amplification for Rapid Detection ofNewcastle Disease Virusrdquo Journal of Clinical Microbiology vol43 no 4 pp 1646ndash1650 2005

[95] H Kirunda O M M Thekisoe P D Kasaija et al ldquoUse ofreverse transcriptase loop-mediated isothermal amplificationassay for field detection of newcastle disease virus using lessinvasive samplesrdquo Veterinary World vol 5 no 4 pp 206ndash2122012

[96] M J Heller ldquoDNA microarray technology devices systemsand applicationsrdquoAnnual Review of Biomedical Engineering vol4 no 1 pp 129ndash153 2002

[97] D M Dankbar E D Dawson MMehlmann et al ldquoDiagnosticmicroarray for influenza B virusesrdquo Analytical Chemistry vol79 no 5 pp 2084ndash2090 2007

[98] X Han X Lin B Liu et al ldquoSimultaneously subtyping ofall influenza A viruses using DNA microarraysrdquo Journal ofVirological Methods vol 152 no 1-2 pp 117ndash121 2008

[99] O Lung A Beeston S Ohene-Adjei et al ldquoElectronic microar-ray assays for avian influenza and Newcastle disease virusrdquoJournal of Virological Methods vol 185 no 2 pp 244ndash253 2012

[100] L-C Wang C-H Pan L L Severinghaus et al ldquoSimultaneousdetection and differentiation of Newcastle disease and avianinfluenza viruses using oligonucleotide microarraysrdquo Veteri-nary Microbiology vol 127 no 3-4 pp 217ndash226 2008

[101] N Laamiri P Fallgren S Zohari et al ldquoAccurate detectionof avian respiratory viruses by use of multiplex PCR-basedluminex suspensionmicroarray assayrdquo Journal ofClinicalMicro-biology vol 54 no 11 pp 2716ndash2725 2016

[102] K T Sultankulova N S Kozhabergenov V M Strochkov et alldquoNew oligonucleotide microarray for rapid diagnosis of avianviral diseasesrdquoVirology Journal vol 14 no 1 article no 69 2017

[103] J P Chambers B P Arulanandam L L Matta A Weis andJ J Valdes ldquoBiosensor recognition elementsrdquo Current Issues inMolecular Biology vol 10 no 1 pp 1ndash12 2008

[104] M L Sin K E Mach P KWong and J C Liao ldquoAdvances andchallenges in biosensor-based diagnosis of infectious diseasesrdquoExpert Review of Molecular Diagnostics vol 14 no 2 pp 225ndash244 2014

[105] S Sang Y Wang Q Feng Y Wei J Ji andW Zhang ldquoProgressof new label-free techniques for biosensors A reviewrdquo CriticalReviews in Biotechnology vol 36 no 3 pp 465ndash481 2016

[106] F C Dudak and I H Boyaci ldquoRapid and label-free bacteriadetection by surface plasmon resonance (SPR) biosensorsrdquoBiotechnology Journal vol 4 no 7 pp 1003ndash1011 2009

[107] A D Taylor J Ladd Q Yu S Chen J Homola and S JiangldquoQuantitative and simultaneous detection of four foodbornebacterial pathogens with a multi-channel SPR sensorrdquo Biosen-sors and Bioelectronics vol 22 no 5 pp 752ndash758 2006

[108] L Shi Q Sun J He et al ldquoDevelopment of SPR biosensor forsimultaneous detection of multiplex respiratory virusesrdquo Bio-Medical Materials and Engineering vol 26 pp S2207ndashS22162015

[109] H Bai R Wang B Hargis H Lu and Y Li ldquoA SPR aptasensorfor detection of avian influenza virusH5N1rdquo Sensors vol 12 no9 pp 12506ndash12518 2012

[110] C D Kang S W Lee T H Park and S J Sim ldquoPerformanceenhancement of real-time detection of protozoan parasiteCryptosporidium oocyst by a modified surface plasmon reso-nance (SPR) biosensorrdquo Enzyme and Microbial Technology vol39 no 3 pp 387ndash390 2006

[111] B Luo Y Xu S Wu et al ldquoA novel immunosensor based onexcessively tilted fiber grating coated with gold nanospheresimproves the detection limit ofNewcastle disease virusrdquoBiosen-sors and Bioelectronics vol 100 pp 169ndash175 2018

[112] R H Deurenberg E BathoornM A Chlebowicz et al ldquoAppli-cation of next generation sequencing in clinical microbiologyand infection preventionrdquo Journal of Biotechnology vol 243 pp16ndash24 2017

[113] M LMetzker ldquoSequencing technologiesmdashthe next generationrdquoNature Reviews Genetics vol 11 no 1 pp 31ndash46 2010

[114] S Souf ldquoRecent advances in diagnostic testing for viral infec-tionsrdquo Bioscience Horizons vol 9 no 1 2016

[115] Y He T L Taylor K M Dimitrov et al ldquoWhole-genomesequencing of genotype VI Newcastle disease viruses fromformalin-fixed paraffin-embedded tissues from wild pigeonsreveals continuous evolution and previously unrecognizedgenetic diversity in theUSrdquoVirology Journal vol 15 no 1 2018

[116] K M Dimitrov P Sharma J D Volkening et al ldquoA robustand cost-effective approach to sequence and analyze completegenomes of small RNA virusesrdquo Virology Journal vol 14 no 1article no 72 2017

[117] C De Battisti A Salomoni S Ormelli I Monne I Capua andG Cattoli ldquoRapid pathotyping of Newcastle Disease Virus bypyrosequencingrdquo Journal of Virological Methods vol 188 no 1-2 pp 13ndash20 2013

[118] D K Byarugaba K K Mugimba J B Omony et al ldquoHighpathogenicity and low genetic evolution of avian paramyx-ovirus type i (Newcastle disease virus) isolated from live birdmarkets in Ugandardquo Virology Journal vol 11 no 1 article no173 2014

[119] A Djikeng R Halpin R Kuzmickas et al ldquoViral genomesequencing by random priming methodsrdquo BMC Genomics vol9 article no 5 2008

[120] T Rosseel M Scheuch D Hoper et al ldquoDNase SISPA-next generation sequencing confirms Schmallenberg virus inBelgian field samples and identifies genetic variation in EuroperdquoPLoS ONE vol 7 no 7 Article ID e41967 2012

[121] S Kumar R Kumari and V Hallan ldquoSequence-independentAmplification with Genome Multiplexing to Establish Com-plete Genome of Multipartite RNA Viruses Cucumber mosaicvirus as a Case Studyrdquo Journal of Phytopathology vol 165 no 6pp 361ndash366 2017

[122] A R Macalalad M C Zody P Charlebois et al ldquoHighlysensitive and specific detection of rare variants in mixedviral populations from massively parallel sequence datardquo PLoSComputational Biology vol 8 no 3 Article ID e1002417 2012

[123] K Chrzastek D-H Lee D Smith et al ldquoUse of Sequence-Independent Single-Primer-Amplification (SISPA) for rapiddetection identification and characterization of avian RNAvirusesrdquoVirology vol 509 pp 159ndash166 2017

[124] A Conan F L Goutard S Sorn and S Vong ldquoBiosecuritymeasures for backyard poultry in developing countries asystematic reviewrdquo BMC Veterinary Research vol 8 article 2402012

[125] S B Hitchner G Reising and H Van Roekel ldquoCharacteristicsof the B1 strain of Newcastle disease virusrdquoAmerican Journal ofVeterinary Research vol 12 no 44 pp 246ndash249 1951


[126] RWWinterfield C L Goldman andEH Seadale ldquoNewcastleDisease Immunization Studies 4 Vaccination of Chickenswith B1 F and Lasota Strains of Newcastle Disease VirusAdministered through theDrinkingWaterrdquoPoultry Science vol36 no 5 pp 1076ndash1088 1957

[127] Z Bensink and P Spradbrow ldquoNewcastle disease virus strainI2 - A prospective thermostable vaccine for use in developingcountriesrdquo Veterinary Microbiology vol 68 no 1-2 pp 131ndash1391999

[128] D A Senne D J King and D R Kapczynski ldquoControl ofNewcastle disease by vaccinationrdquo Developmental Biology vol119 pp 165ndash170 2004

[129] F Rauw Y Gardin V Palya et al ldquoHumoral cell-mediated andmucosal immunity induced by oculo-nasal vaccination of one-day-old SPF and conventional layer chicks with two differentlive Newcastle disease vaccinesrdquo Vaccine vol 27 no 27 pp3631ndash3642 2009

[130] F Perozo P Villegas R Dolz C L Afonso and L B PurvisldquoThe VGGA strain of Newcastle disease virus Mucosal immu-nity protection against lethal challenge andmolecular analysisrdquoAvian Pathology vol 37 no 3 pp 237ndash245 2008

[131] E D de Geus J M J Rebel and L Vervelde ldquoInductionof respiratory immune responses in the chicken implicationsfor development of mucosal avian influenza virus vaccinesrdquoVeterinary Quarterly vol 32 no 2 pp 75ndash86 2012

[132] D H Thornton I G Hopkins and C Nancy Hebert ldquoPotencyof live newcastle disease vaccinesrdquo Avian Pathology vol 9 no3 pp 457ndash464 1980

[133] R W Winterfield A S Dhillon and L J Alby ldquoVaccinationof chickens against Newcastle disease with live and inactivatedNewcastle disease virusrdquoPoultry Science vol 59 no 2 pp 240ndash246 1980

[134] J L Tlaxca S Ellis and R L Remmele ldquoLive attenuatedand inactivated viral vaccine formulation and nasal deliveryPotential and challengesrdquoAdvancedDrug Delivery Reviews vol93 pp 56ndash78 2014

[135] N Razmaraii R Toroghi H Babaei I Khalili S Sadigh-Eteghad and L Froghy ldquoImmunogenicity of commercialformaldehyde and binary ethylenimine inactivated Newcastledisease virus vaccines in specific pathogen free chickensrdquoArchives of Razi Institute vol 67 no 1 pp 21ndash25 2012

[136] S-I Fukanoki T Iwakura S Iwaki et al ldquoSafety and efficacyof water-in-oil-in-water emulsion vaccines containing Newcas-tle disease virus haemagglutinin-neuraminidase glycoproteinrdquoAvian Pathology vol 30 no 5 pp 509ndash516 2001

[137] L Zhai Y Li W Wang and S Hu ldquoEnhancement of humoralimmune responses to inactivated Newcastle disease and avianinfluenza vaccines by oral administration of ginseng stem-and-leaf saponins in chickensrdquo Poultry Science vol 90 no 9 pp1955ndash1959 2011

[138] NADoria-Rose andN LHaigwood ldquoDNAvaccine strategiesCandidates for immune modulation and immunization regi-mensrdquoMethods vol 31 no 3 pp 207ndash216 2003

[139] M Firouzamandi H Moeini D Hosseini et al ldquoImprovedimmunogenicity of Newcastle disease virus fusion proteingenesrdquo Journal of Veterinary Science vol 17 no 1 pp 21ndash262016

[140] M Firouzamandi HMoeini S D Hosseini et al ldquoPreparationcharacterization and in ovo vaccination of dextran-sperminenanoparticle dna vaccine coexpressing the fusion and hemag-glutinin genes against newcastle diseaserdquo International Journalof Nanomedicine vol 11 pp 259ndash267 2016

[141] K Zhao Y Zhang X Zhang et al ldquoPreparation and efficacyof newcastle disease virus dna vaccine encapsulated in chitosannanoparticlesrdquo International Journal ofNanomedicine vol 9 no1 pp 389ndash402 2014

[142] K Zhao X Duan L Hao X Wang and Y Wang ldquoImmuneeffect of newcastle disease virus DNA vaccine with c3d as amolecular adjuvantrdquo Journal of Microbiology and Biotechnologyvol 27 no 11 pp 2060ndash2069 2017

[143] K Zhao J Han Y Zhang et al ldquoEnhancing mucosal immuneresponse of Newcastle disease virus DNA vaccine using N-2-hydroxypropyl trimethyl ammonium chloride chitosan and NO-carboxymethyl chitosan nanoparticles as delivery carrierrdquoMolecular Pharmaceutics vol 15 no 1 pp 226ndash237 2017

[144] S C Weli and M Tryland ldquoAvipoxviruses Infection biologyand their use as vaccine vectorsrdquo Virology Journal vol 8 no1 article no 49 2011

[145] Y Li K Reddy S M Reid et al ldquoRecombinant herpesvirusof turkeys as a vector-based vaccine against highly pathogenicH7N1 avian influenza andMarekrsquos diseaserdquo Vaccine vol 29 no46 pp 8257ndash8266 2011

[146] G Meulemans C Letellier M Gonze M C Carlier and ABurny ldquoNewcastle disease virus f glycoprotein expressed froma recombinant vaccinia virus vector protects chickens againstlive-virus challengerdquoAvian Pathology vol 17 no 4 pp 821ndash8271988

[147] K J Ewer T Lambe C S Rollier A J Spencer A V SHill and L Dorrell ldquoViral vectors as vaccine platforms Fromimmunogenicity to impactrdquo Current Opinion in Immunologyvol 41 pp 47ndash54 2016

[148] K Karaca J M Sharma B J Winslow et al ldquoRecombinantfowlpox viruses coexpressing chicken type I IFN and newcastledisease virus HN and F genes Influence of IFN on protectiveefficacy and humoral responses of chickens following in ovo orpost-hatch administration of recombinant virusesrdquoVaccine vol16 no 16 pp 1496ndash1503 1998

[149] H L Sun Y F Wang D Y Miao P J Zhang H D Zhi L LXu et al ldquoConstruction and characterization of recombinantfowlpox virus co-expressing F and HN genes of newcastledisease virus and gB gene of infectious larygnotracheitis virusrdquoChinese Journal of Biotechnology vol 22 no 6 pp 931ndash9382006

[150] M Esaki A Godoy J K Rosenberger et al ldquoProtectionand antibody response caused by turkey herpesvirus vectornewcastle disease vaccinerdquo Avian Diseases vol 57 no 4 pp750ndash755 2013

[151] R W Morgan J Gelb Jnr C R Pope and P J A SondermeijerldquoEfficacy in chickens of a herpesvirus of turkeys recombinantvaccine containing the fusion gene of Newcastle disease virusOnset of protection and effect of maternal antibodiesrdquo AvianDiseases vol 37 no 4 pp 1032ndash1040 1993

[152] V Palya I Kiss T Tatar-Kis T Mato B Felfoldi and YGardin ldquoAdvancement in vaccination against newcastle diseaseRecombinant HVT NDV provides high clinical protection andreduces challenge virus shedding with the absence of vaccinereactionsrdquo Avian Diseases vol 56 no 2 pp 282ndash287 2012

[153] S-H Kim S Xiao H Shive P L Collins and S KSamal ldquoReplication neurotropism and pathogenicity of avianparamyxovirus serotypes 1-9 in chickens andducksrdquoPLoSONEvol 7 no 4 Article ID e34927 2012

[154] S Kumar B Nayak P L Collins and S K Samal ldquoEvaluationof the newcastle disease virus f and hn proteins in protectiveimmunity by using a recombinant avian paramyxovirus type 3


vector in chickensrdquo Journal of Virology vol 85 no 13 pp 6521ndash6534 2011

[155] N Kushnir S J Streatfield and V Yusibov ldquoVirus-like particlesas a highly efficient vaccine platform diversity of targets andproduction systems and advances in clinical developmentrdquoVaccine vol 31 no 1 pp 58ndash83 2012

[156] M O Mohsen L Zha G Cabral-Miranda and M F Bach-mann ldquoMajor findings and recent advances in virusndashlikeparticle (VLP)-based vaccinesrdquo Seminars in Immunology vol34 pp 123ndash132 2017

[157] H D Pantua L W McGinnes M E Peeples and T G Morri-son ldquoRequirements for the assembly and release of Newcastledisease virus-like particlesrdquo Journal of Virology vol 80 no 22pp 11062ndash11073 2006

[158] J-K Park D-H Lee S-S Yuk et al ldquoVirus-like particle vaccineconfers protection against a lethal newcastle disease virus chal-lenge in chickens andallows a strategy of differentiating infectedfrom vaccinated animalsrdquo Clinical and Vaccine Immunologyvol 21 no 3 pp 360ndash365 2014

[159] L W McGinnes H Pantua J P Laliberte K A Gravel S Jainand T G Morrison ldquoAssembly and biological and immunolog-ical properties of Newcastle disease virus-like particlesrdquo Journalof Virology vol 84 no 9 pp 4513ndash4523 2010

[160] T G Morrison ldquoNewcastle disease virus-like particles as aplatform for the development of vaccines for human andagricultural pathogensrdquo Future Virology vol 5 no 5 pp 545ndash554 2010

[161] C K Pfaller R Cattaneo and M J Schnell ldquoReverse geneticsof Mononegavirales How they work new vaccines and newcancer therapeuticsrdquoVirology pp 1ndash14 2015

[162] K Gururaj J J Kirubaharan V K Gupta and R S PawaiyaldquoReview article past and present of reverse genetics in animalvirology with special reference to non-segmented negativestranded rna viruses a reviewrdquo Advances in Animal and Veteri-nary Sciences vol 2 pp 40ndash48 2014

[163] S Xiao B Nayak A Samuel et al ldquoGeneration by ReverseGenetics of an Effective Stable Live-Attenuated NewcastleDisease VirusVaccineBased on a CurrentlyCirculatingHighlyVirulent Indonesian Strainrdquo PLoS ONE vol 7 no 12 Article IDe52751 2012

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these proteins the F is generally considered to be amolecularmarker of NDV virulence According to the OIE virulentNDV strains are diagnosed as those possessing multiple basicamino acid residues (arginine and lysine) at the F proteincleavage site located between amino acid positions 112-116and a phenylalanine residue at position 117 On the otherhand isolates of low virulence are considered to be those withmonobasic F cleavage site and a leucine residue at position117 [2] Unfortunately this is not a universal rule of thumbas some NDV strains adapted in wild birds such as pigeonsand doves have been shown to be minimally pathogenicdespite possessing the so-called polybasic F cleavage site [45] Furthermore some of these pigeon paramyxoviruses havebeen reported to manifest a dramatic increase in virulencefollowing a few passages in chicken without the accompany-ing nucleotide changes in the entire F coding region [6 7] Inaddition swapping the F genes of avirulent pigeon adaptedNDV with that of a virulent NDV strain or vice versa didnot lead to the change in the virulence of the generatedchimeric viruses These observations altogether signify thatother factors independent of the F cleavage site are importantin determining the virulence of NDV in chicken Indeedthe HN and L proteins have recently been proven to bedirectly associated with NDV virulence [8 9] Hence thebiggest diagnostic dilemma is whether targeted evaluationof F cleavage site is still a reliable molecular indicator ofNDV virulence or complete genome analysis is required toadequately predict the virulence potential of NDV isolates

Epidemiological evidences indicate that NDV is con-stantly evolving and its isolates are so far classified intomore than eighteen phylogenetically distinct genotypes [14]Regardless of their genetic variability however all NDVisolates are serologically grouped into a single serotype calledavian paramyxovirus type-1 [15] This implies that immunityinduced by any NDV strain should be able to cross-protectagainst challenge with any other virulent strain because ofthe fairly similar antigenic properties shared by all NDVisolates Indeed for over sixty years both live attenuatedand inactivated NDV vaccines have been extensively used tocurb the economic menace of ND across the globe [16] Inparticular the live vaccines are known for their track recordof high efficacy due to their ability to efficiently replicateand induce a robust immune response following a singleadministration They are also suitable formass application viaspray or drinking water [17] Unfortunately they are unableto block the replication of the heterologous virulent NDVdespite protecting against overt clinical disease [18] As aresult birds vaccinated with those vaccines may serve asreservoirs for virulentNDV shedding a substantial amount ofthe infectious virus into the environment leading to potentialoutbreaks among the nonprotected in-contact birds [19]These shortcomings of the conventional vaccines collectivelycall for the need to improve the current vaccination strategiesagainst the prevalent NDV strains across the globe

Recently the global poultry industry has witnessed anupsurge in the emergence of cutting-edge techniques for theidentification and control of virulent NDV infection Thesestate-of-the-art technologies have no doubt demonstrated thepotentials to overcome the loopholes of their conventional

counterparts Here we comprehensively review the currentand next-generation diagnostic and vaccination strategies forNDV with special emphasis on the strengths and weaknessesof each of those techniques

2 Etiology

21 Classification of NDV Isolates of NDV are membersof the genus Avulavirus in the family Paramyxoviridae Atpresent a total of 15 avian paramyxovirus serotypes (APMV-1to APMV-15) have been identified in different species of wildand domesticated birds where they may be associated withrespiratory disease and a drastic reduction in egg production[20] Members of APMV-1 made up of NDV isolates are themost economically important and are genetically classifiedinto two broad groups class I and class II Whereas membersof class I isolates are grouped into only one genotype isolatesbelonging to class II are further subdivided into genotypes I-XVIII which are all predicted to be virulent in chicken exceptsome isolates in genotypes I II and X [21 22]

22 Molecular Biology of NDV The genetic material ofNDV is a negative stranded nonsegmented RNA that strictlyadheres to the rule of six (genome size divisible by six)having a total size of 15186 15192 or 15198 bp [23] Thegenome houses six genes namely nucleocapsid protein (NP)phosphoprotein (P) matrix protein (M) fusion protein (F)hemagglutinin-neuraminidase protein (HN) and the largeprotein (L) Apart from the P gene which can be transcribedinto three different mRNAs encoding one structural (P)and two nonstructural (V and W) proteins [24] all otherviral genes are monocistronic encoding a single structuralprotein The 31015840 and 51015840 ends of the viral genome constitute theleader and trailer regions which accommodate the regulatorysignals for virus transcription and replication [25]

Morphologically NDV appears to be a pleomorphicenveloped particle with projections of F and HN spike gly-coproteins (Figure 1) that participate in the initiation of virusinfectious cycle (Sachin et al 2011) Immediately beneath theviral envelope is the M protein which is known to maintainthe shape of the virus and assist in the packaging and releaseof the newly assembled viruses [26] The other three proteinsare intimately associated with the viral genome and areknown to perform replication related functions In particularthe NP precisely covers the entire viral RNA to form theribonucleoprotein (RNP) which is the minimum templaterequired for virus replication and mRNA biosynthesis [27]The L protein is the RNA dependent RNA polymerase thatserves as the viral replicase and transcriptase during theinfectious cycle while the P protein is the cofactor of thepolymerase [8]

23 Epidemiology and Emergence of the Virulent NDV Inthe last nine decades four major economically devastatingNDV panzootics have occurred The first one which begansimultaneously in Asia and Europe around the mid-1920swas spreading to the rest of the world rather slowly andtook about twenty years to become fully established [28]Thesecond pandemic however became full pledged within four


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riewarticle - hindawimar 23, 2018 · biomedresearchinternational hf969184.1 chicke/i or c...

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