Characterisation Of Avian Nephritis Virus (ANV) In Australian .

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Characterisation of aviannephritis virus (ANV) inAustralian commercialpoultryAPRIL 2015RIRDC Publication No. 15/049

Characterisation of aviannephritis virus (ANV) inAustralian commercial poultryby Kylie Hewson, Anthony Chamings, Denise O’Rourke, Khrisdiana Putri,Nadeeka Wawegama, Amir Noormohammadi and Jagoda IgnjatovicApril 2015RIRDC Publication No 15/049RIRDC Project No PRJ-005199

2015 Rural Industries Research and Development Corporation.All rights reserved.ISBN 978-1-74254-795-4ISSN 1440-6845Characterisation of avian nephritis virus (ANV) in Australian commercial poultryPublication No. 15/049Project No. PRJ-005199The information contained in this publication is intended for general use to assist public knowledge and discussionand to help improve the development of sustainable regions. You must not rely on any information contained inthis publication without taking specialist advice relevant to your particular circumstances.While reasonable care has been taken in preparing this publication to ensure that information is true and correct,the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication.The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), theauthors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liabilityto any person, arising directly or indirectly from any act or omission, or for any consequences of any such act oromission, made in reliance on the contents of this publication, whether or not caused by any negligence on thepart of the Commonwealth of Australia, RIRDC, the authors or contributors.The Commonwealth of Australia does not necessarily endorse the views in this publication.This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights arereserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rightsshould be addressed to RIRDC Communications on phone 02 6271 4100.Researcher Contact DetailsJagoda IgnjatovicThe University of MelbourneSchool of Veterinary Science250 Princes HwyWerribee VIC 3030Phone: 03 9731 2265Fax:03 9731 2366Email: submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.RIRDC Contact DetailsRural Industries Research and Development CorporationLevel 2, 15 National CircuitBARTON ACT 2600PO Box 4776KINGSTON ACT 2604Phone:Fax:Email:Web:02 6271 410002 6271 tronically published by RIRDC in April 2015Print-on-demand by Union Offset Printing, Canberra at phone 1300 634 313Front cover image from Protein structure prediction on the web: a case study using the Phyre server, Kelley LAand Sternberg MJE. Nature Protocols 4, 363 - 371 (2009).ii

ForewordAvian nephritis virus (ANV) has been circulating in poultry flocks in several countries since at least1979 but was detected in Australian poultry recently in 2010. It was important to characterise theAustralian strains of ANV and establish diagnostic tests to monitor its presence and spread inAustralian poultry.The aim of this project was to identify and develop suitable diagnostic tests to be used to monitor thisvirus. Prior to this investigation, there was no established diagnostic test, serological or molecular, forthe detection of Australian strains of ANV. International efforts have focussed on the development ofmolecular tests due to difficulties in obtaining in vitro-adapted viruses for use in serological assays.Little is known about the clinical situation of ANV in poultry flocks, and development of diagnostictests would help to establish the breadth of ANV infection. ANV has been known to be associated withboth subclinical and clinical infections, suggesting the possible presence of virulent and non-virulentstrains of the virus. Also, specific clinical situations such as heat stress or co-infectious pathogenscould contribute to the clinical impact of ANV infections.The research described in this report benefits the Australian poultry industry as it provides furtherinformation regarding viruses that potentially have a negative economic impact on poultry production.This project identified many of the key characteristics of ANV, as well as a novel avian reovirus(ARV), which set foundations to develop additional diagnostic tests and control strategies. Furtherinvestigation into the epidemiology, diversity and virulence of these strains would aid in determining abetter estimation of the economic impact of these viruses on the Australian poultry industry.This report is an addition to RIRDC’s diverse range of over 2000 research publications and it formspart of our Chicken Meat R&D program, which aims to stimulate and promote R&D that will deliver aproductive and sustainable Australian chicken meat industry that provides quality wholesome food tothe nation.Most of RIRDC’s publications are available for viewing, free downloading or purchasing online Purchases can also be made by phoning 1300 634 313.Craig BurnsManaging DirectorRural Industries Research and Development Corporationiii

AcknowledgmentsThe investigators would like to acknowledge contributions by Dr Soy Rubite, Dr Ben Wells and DrJohn Tchang through submission of clinical specimens and relevant flock history. Also Cheryl Colsonand June Daly are thanked for their assistance with the in vivo experiments. We especiallyacknowledge the generous assistance of the entire staff from the Laboratory of Associate Professor A.Hill (Bio21, Melbourne) with the Ion Torrent sequencing techniques and use of relevant equipment.We would also like to acknowledge the productive discussions with Dr. Daniel Todd (Queen'sUniversity of Belfast, UK) as well as the assistance of Dr Annette Bolte-Terberger (Lohmann,Cuxhaven Germany) for analysing our ANV sera in their ANV assays.AbbreviationsAEVavian encephalomyelitis virusAIVavian influenza virusANVavian nephritis virusARVavian reovirusBLSbig liver and spleen diseasebpbase pair/sCAMchorioallantoic membraneCAVchicken anaemia virusCEKchicken embryo kidneyCPEcytopathic effectDEPCdiethylpyrocarbonateDMEMDulbecco's Modified Eagle MediumELISAenzyme-linked immuno-sorbent assayELVenteric-like virusEMelectron microscopyFAVfowl adenovirusHAstVhuman AstrovirusHRMhigh resolution meltHVTherpes virus of turkeysIFAimmuno-fluorescence assayIBDinfectious bursal diseaseLMHchicken hepatocellular carcinoma epithelial cell lineMAbmonoclonal antibodyMDVMaerek’s disease virusNDVNewcastle’s disease virusORFopen reading framePBSphosphate buffered salinePIpost inoculationSPFspecific pathogen freeVTMviral transport mediaiv

ContentsForeword . iiiAcknowledgments . ivAbbreviations . ivExecutive Summary. ixIntroduction . 13Objectives . 16Methodology. 17Specimen history . 17Virus preparation . 18Determination of viral molecular characteristics . 18Propagation and isolation of ANV . 21ANV Serological characterisation and ELISA development . 24Identification of the unknown contaminating virus . 261. Molecular characterisation of Australian ANVs . 28Sequence analysis demonstrated the diversity of Australian ANVs . 28Phylogenetic analysis demonstrated the diversity of Australian ANV strains . 31Recombination analysis identified recombination had occurred in Australian ANVs . 312. Development of molecular tools for ANV detection and strain identification . 33The original ANVPoly PCR was found to amplify too many non-specific products . 33A HRM curve analysis technique was developed for the differentiation of ANV. 35Differentiation of ANV strains using the ANVCap PCR was promising based on ampliconsequencing. 37Differentiation of ANV strains by ANVCap PCR/HRM was confirmed by entire capsidprotein gene sequencing . 37Field application of the ANVCap PCR/HRM technique was successful in detecting ANV . 393. Isolation of ANV and production of mono-specific antisera . 40Time-Course analysis of ANV growth in vitro by PCR suggested that the CEK cells werenot permissive for ANV propagation . 40Australian ANVs were not able to be propagated in CEK cells . 40Propagation in other cells in vitro was unsuccessful . 41Propagation in ovo was unsuccessful . 41Propagation in vivo was successful for isolation of pure ANV and subsequent productionof ANV mono-specific antisera . 41ANV viral stocks were confirmed to be similar to ANV present in the diagnosticspecimens . 42Reference ANV antisera was produced . 43Australian ANV strains did not cross-react with international strain ANV1 in the currentlyavailable test. 43v

Summary of what was obtained for each clinical specimen . 434. Development of ELISA for detection of antibodies to ANV . 44Generation of antigens for use in an ANV antibody ELISA . 44Evaluation of the specificity of the NSW 3 rp27 ELISA and reference ANV antisera . 47Four reference ANV antisera were free from antibodies to all other known avianpathogens . 48ELISA based on the NSW 3 rp27 is a highly sensitive test and able to detect antibodies toANV1 and ANV2 strains . 49Standardisation of the NSW 3 rp27 ELISA . 52Testing of known ANV positive field sera using the NSW 3 rp27 ELISA was successful . 55No antibodies to ANV were detected in an SPF flock using the NSW 3 rp27 ELISA . 55The NSW 3 rp27 ELISA was shown to be sensitive and specific for ANV . 55Optimised methodology for the NSW 3 rp27 ANV ELISA . 565. Isolation and characterisation of the novel avian reovirus (ARV). 57Growth characteristics of the unknown virus. 57Serological assessment of the unknown virus determined that it was a novel virus . 57The novel virus was found to be a small RNA virus . 58Molecular characterisation of the unknown virus determined it to be a novel ARV . 58A PCR for the detection of the novel ARV was developed . 59The novel ARVs were 85 – 87 % similar to the RAM1 reference ARV strain . 60Phylogenetic analysis of the novel ARVs suggested diversity . 62Serological characterisation of the novel ARVs . 63Virus stocks of the novel ARVs were produced . 63Results. 64ANV is prevalent in Australian broiler flocks . 64Growth properties of Australian ANV in vitro differed from reference strain ANV1. 64Genetic analysis of Australian ANV strains demonstrated their diversity from internationalstrains . 65Methods for detection and differentiation of ANV strains were developed . 66A sensitive and specific ELISA for the detection of ANV antibodies was developed . 66A novel ARV was isolated, propagated and successful diagnostic assays were developed . 67Implications . 68Recommendations. 69References . 70vi

TablesTable 1.Designation and origin of ANV strains used in the study . 17Table 2.ORF1b partial protein sequence identities between Australian ANV strains andinternational reference strains ANV1 and ANV2 . 28Table 3.Similarity between the capsid amino acid sequence of Australian ANV strain withinternational reference groups . 29Table 4.Percentage similarity of the ANVCap PCR amplicon sequences from molecular clonedviruses . 37Table 5.The sequence similarity of the entire capsid protein amino acid sequence of molecularclones from Australian ANV specimens compared to international strains . 38Table 6.Prevalence of ANV in broiler flocks aged 5 to 23 days from one Queensland poultrycompany . 39Table 7Summary of what was obtained for each original clinical specimen . 43Table 8.Efficacy of various antigens derived from NSW 3 ANV for detection of ANV antibodiesin ELISA . 46Table 9.Comparison of antigens derived from NSW 3 to detect antibodies to all Australian ANVstrains . 47Table 10.Reaction of antisera to all known avian pathogens in the NSW 3 rp27 ELISA . 48Table 11.Testing of reference ANV sera for absence of antibodies to other avian pathogens . 49Table 12.Endpoint titres of reference ANV antisera using rp27 from different ANV strains asantigens in ELISA . 51Table 13.The ELISA and Western blotting results from 213 sera to determine the sensitivity andspecificity of the NSW 3 rp27 ELISA . 56Table 14.Comparison of the partial nucleotide sequences of the L1 segment of four isolated novelARVs . 61FiguresFigure 1The genomic organisation of ANV . 19Figure 2Location of nine antigenic sites/peptides in the p27 fragment of NSW 3 capsid protein . 25Figure 3Location of antigenic sites/peptides in the p34 fragment of NSW 3 capsid protein. 25Figure 4International ANV groupings as determined by Todd et al 2011 based on the capsidprotein sequence. 30Figure 5.SimPlot (recombination) analysis of VIC 3 (upper panel) and NSW 4 (lower panel).Similarity is shown on a scale from 0.5 (50 %) to 1.0 (100 %) . 32Figure 6.An example of the results obtained using the ANVPoly PCR for the detection of ANV . 33Figure 7.An example of the results obtained using the ANVCap PCR for the detection of ANV . 34Figure 8.An example of the results obtained using the ANVentireCap PCR for the detection ofANV . 34Figure 9.Differentiation of Australian ANV strains using the HRM method based on the 3’ end ofANV genome (ANVCap PCR) . 35vii

Figure 10.An example of the differentiation of molecular cloned Australian ANV strains using theHRM PCR technique based on the ANV ORF1b region . 36Figure 11.An example of the differentiation of molecular cloned Australian ANV strains using theHRM PCR technique based on the ANV capsid-3'UTR region . 36Figure 12Time-course analysis of ANV in CEK cells. . 40Figure 13Isolation of ANV from chickens infected for antisera production. . 42Figure 14.Expression and purification of NSW 3 and NSW 4 capsid protein . 45Figure 15.Expression in Baculovirus of rp27 and rp34 derived from NSW 3 strain . 45Figure 16.NSW 3 rp27 antigen reacted equally well with antibodies to all Australian ANV strainsand was less cross-reactive with ANV 1 antibodies . 50Figure 17.ANV1 rp27 antigen was less cross-reactive and not able to detect antibodies to threeAustralian ANV strains in sera at dilution 1/400 . 50Figure 18.ANV 2 rp27 antigen is cross-reactive and also able to detect antibodies to all AustralianANV strains . 51Figure 19.Influence of dilution buffer on the binding of 51 individual reference ANV sera at adilution of 1/400 to NSW 3 rp27 antigen . 52Figure 20.Influence of dilution buffer on binding of 68 individual SPF sera at 1/400 dilution to rp27NSW 3 antigen . 53Figure 21.S/P ratio of 58 ANV reference sera at 1/400 dilution on NSW 3 rp27 antigen . 54Figure 22.S/P ratio for 68 SPF sera tested at 1/400 dilution on NSW 3 rp27 antigen . 54Figure 23.S/P ratio of 55 field sera from flocks from which NSW 1, NSW 2, NSW 3 and VIC 3were isolated . 55Figure 24CPE observed on CEK cells infected with the unknown virus at 5 days PI . 57Figure 25.Transmission electron microscopy of the novel ARV . 58Figure 26.Genomic organisation of ARV. . 59Figure 27.An example of the results of ARV PCRs used in this study. . 60Figure 28The diversity of avian orthoARVs based on the 3’ sequence of the L1 segment of the viralgenome . 62viii

Executive SummaryWhat the report is aboutThis report describes the characterisation of Australian ANV strains and the development of molecularand serological diagnostic tests. The discovery and initial characterisation of a novel avian reovirus(ARV) are also described.Who is the report targeted at?The information will aid all Australian poultry producers, specifically those growing young chicks( two week old). The diagnostic tools developed in this investigation will assist farmers, producers,poultry veterinarians and vaccine manufacturers to monitor ANV infections/contamination andestimate its impact on production of poultry, and potentially the development of vaccines.BackgroundANV has been reported in poultry flocks worldwide and predominately causes disease in chickens lessthan two-weeks old. Older chickens appear to be less susceptible to the clinical manifestation of thedisease (Gough and McNulty 2008). The first report of ANV was in 1979 in Japan (Yamaguchi, Imadaet al. 1979) however little is known about the ANV in Australia since this virus was first detected inAustralian poultry in 2010 (Hewson, O'Rourke et al. 2010). ANV infection can be clinical orsubclinical. In the former type of infection, clinical signs may include diarrhoea, polyurea,dehydration, growth retardation and mortality. Some of these clinical manifestations can be difficult todistinguish from those induced by nephropathogenic strains of infectious bronchitis viruses (Cumming1999; Ignjatovic, Ashton et al. 2002). There are currently no effective treatment or control strategiesfor ANV infection. Complete cleanout of the shed between flocks may assist but, given that the virusis suspected to shed vertically, does not totally prevent the infection. To date no vaccine has beendeveloped for ANV.Diagnostic tools to detect and characterise ANV have been developed globally, however due todifficulties in adaptation of ANV to growth in cell culture, the use of molecular assays have beenpreferred over serological assays (Pantin-Jackwood, Todd et al. 2013). Serological assays such asELISA have been developed, however all utilise the same Japanese strain (Shirai, Nakamura et al.1990; Decaesstecker and Meulemans 1991; Mockett, Huggins et al. 1993) or a region-specific strain(Zhao, Zhu et al. 2012). Molecular tests, such as PCR, have been performed to confirm the presence ofANV in clinical specimens (Day, Spackman et al. 2007; Smyth, Jewhurst et al. 2010; Todd, Trudgettet al. 2010). However, the use of these molecular tests is complicated by the significant geneticdiversity of ANV which reflects the large repertoire of ANV strains and complexity in serologicaldiagnosis (Imada, Yamaguchi et al. 2000; Todd, Trudgett et al. 2010; Pantin-Jackwood, Todd et al.2013).Aims/objectivesThe main objectives of this project were to: develop diagnostic methods for serological characterisation of ANV develop molecular methods for identification and differentiation of ANV sequence the complete genome of the isolated ANV and compare with international ANV strains characterise biological properties of isolated ANV in chicks and eggs determine the prevalence in broiler flocks from suspected and already identified locationsix

The results of this study are expected to benefit the Australian poultry industry by improving theability to monitor ANV infections and diagnose and characterise new strains of the virus. Ultimately,characterisation of Australian ANV strains will enable the future development of more sensitive assaysand more directed control approaches.Methods usedMultiple molecular and serological methodologies including PCR and high resolution melt (HRM)curve analysis were used in this project for the detection, differential diagnosis and characterisation ofANV strains in diagnostic specimens. Various sequencing techniques (including Ion torrentsequencing) were applied to obtain the entire ANV genome sequence, identify contaminating virusesand identify multiple strains in a single specimen. Electron microscopy and various chemical methodswere used to determine and discriminate the physiochemical properties of ANV and a discoveredavian Reovirus (ARV). ELISA was used for detection of ANV antigens or antibodies and in vivo, inovo and in vitro (cell culture and embryonated chicken eggs) techniques were used in attempts toisolate ANV. Protein synthesis and recombinant protein expression were used for production ofantigens for use in serological assays.Results/key findingsThe key findings of this report were: Five ANV strains were isolated from broiler flocks in Australia and partially characterised at thegenetic and antigenic levels (multiple other strains were identified but are yet to be characterised) 75% of the whole genome of one Australian ANV strain was sequenced which enabled positiveidentification of isolated strains as ANV The sequencing of the capsid gene that codes for the major antigen of ANV showed that allAustralian ANVs identified to date are genetically different from each other and also differ fromANV described in other countries such as the US, Japan and the UK Often more than one genetically distinct ANV was present in a flock Recombination was detected in the genome of the Australian ANV strains which suggests in vivorecombination can occur when multiple ANV strains infect a host A novel ARV was isolated from Australian broilers, distinct from all known ARVs This novel ARV was often detected in broilers infected with ANV Molecular diagnostic tests were developed for the detection of Australian ANV and the novelARV Australian ANVs were unable to grow in vitro and were only able to be propagated in chickens Failure to be grown in vitro impacted on the project progress in terms of ability to determinebiological properties of isolated ANV and their economic importance through controlled in vivoexperiments Antisera to Australian ANVs were produced and compared to antisera to ANV1 strain (importedfrom overseas) in serological assays where antigenic differences were detected A part of the ANV capsid protein, known as p27, from an Australian strain was expressed as arecombinant proteinx

An ELISA based on the p27 from an Australian ANV was developed for the detection of ANVantibodies. Evaluation showed the test was specific, sensitive and detected antibodies to allAustralian ANV strains and also antibodies to reference strain ANV1 The ELISA for the detection of ANV antibodies based on the recombinant p27 from ANV1 wasalso evaluated. The test was not able to detect exposure to all Australian ANV confirming theresults obtained by Lohmann P/L that a serological test based on ANV1 may not be able to detectexposure to all ANV strainsImplications for relevant stakeholders:IndustryANV was shown to be prevalent in Australian broilers, with at least 50% of flocks sampled harbouringANV. This indicated that ANV was endemic on many poultry producing sites. A novel type of ARVwas also detected in most of broiler flocks infected with ANV, indicating frequent mixed infectionsand potentially a co-infectious relationship.Although the presence of ANV in some broiler flocks was considered to be the cause of poor flockperformance, a direct link between clinical disease and isolation of ANV could not be directly made.The strains of ANV identified in Australia differ from strains of ANV isolated in other countries andas a result, methods for AN

tests would help to establish the breadth of ANV infection. ANV has been known to be associated with both subclinical and clinical infections, suggesting the possible presence of virulent and non-virulent strains of the virus. Also, specific clinical situations such as heat stress or co-infectious pathogens

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