Avian leukosis virus sub-group J (ALV-J)

Transcription

1 Avian leukosis virus sub-group J (ALV-J) Developing laboratory technologies for diagnosis in Australia A report for the Rural Industries Research and Development Corporation by T. Bagust, S. Fenton and M. Reddy August 2004 RIRDC Publication No 04/116 RIRDC Project No UM-49A

2 2004 Rural Industries Research and Development Corporation. All rights reserved. ISBN ISSN Avian Leukosis sub-group J (ALV-J): Developing laboratory technologies for diagnosis in Australia. Publication No. 04/116 Project No.UM-49A. The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone Researcher Contact Details T. J. Bagust The University of Melbourne Faculty of Veterinary Science Cnr Park Drive and Flemington Rd. Parkville, Victoria Phone: Fax: trevorjb@truck.its.unimelb.edu.au In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: rirdc@rirdc.gov.au Website: Published in July 2004 Printed on environmentally friendly paper by Canprint ii

3 Foreword The ALV-J subgroup of avian leukosis virus has emerged in recent years as causing tumours and mortalities in week old breeder flocks and, following egg transmission, sub-optimal growth in infected progeny broiler flocks. The presence of ALV-J in Australian breeding flocks, and possibly some broiler lines, was detected during a RIRDC scoping study into causes of sub-optimal productivity. The need was identified in the course of this process to develop Australia's technological and research capacity to detect and control ALV-J. The main objectives of this project were to introduce and develop technologies for the culture and detection of ALV-J for the Australian poultry industry and to undertake a comparison of isolates antigenically. The project also sought to develop practical methodologies for diagnosis of ALV-J related field problems in broiler flocks. This report describes the virological, serological and molecular biological techniques developed, assessed and used during this project. The process of selecting the optimum cell types for culture and the sample types that gave the most sensitive detection is described. Selected data is also presented on the prevalence of ALV-J infection in Australian chicken flocks. A small number of selected ALV- J isolates were antigenically characterised and found to differ from other Australian isolates and from overseas strains. Experimental infection studies using two Australian isolates are also described. This project was funded from industry revenue, which is matched, by funds provided by the Australian Government. This report, an addition to RIRDC s diverse range of over 1000 research publications, forms part of our Chicken Meat R & D program, which aims, through carefully focussed R & D, to support increased sustainability and profitability in the chicken meat industry. Most of our publications are available for viewing, downloading or purchasing online through our website: downloads at purchases at Simon Hearn Managing Director Rural Industries Research and Development Corporation iii

4 Acknowledgments We are grateful to all those who assisted with this work including Ms D O Rourke, Ms M Barraza and Mr P Cowling of the International Avian Health Laboratory, University of Melbourne. We also thank Dr Gordon Firth for supplying the original Australian isolate of ALV-J (J98290/191), Dr Guillermo Zavala for supplying UK and US prototypes of ALV-J and antiserum for ADOL-Hc1. We also acknowledge our collaborators from the Australian poultry industry and commercial breeding companies as well as all those industry veterinarians that supplied samples. iv

5 Abbreviations A+/- presence or absence of antibody Ab antibody ADOL-Hc1 ALV-J Hc1 isolate - the USA prototype AGRF Australian Genomic Research Facility ALV avian leukosis virus (following letter indicates the sub-groups eg. ALV-J) ANGIS Australian National Genomic Investigation Service bc buffy-coat preparation bp base pair C/O CEF s primary chicken embryo fibroblasts (C/O propagates all ALV sub-groups) DF-1 transformed chicken fibroblast cell line (C/E propagates all ALV sub-groups except ALV-E) DMEM Dulbecco's Modified Eagle Medium do day old ELISA enzyme linked immunosorbent assay env envelope gene GGP great grand parent GP grand parent gs group specific HPRS-103 Houghton Poultry Research Station HPRS-103 (the original ALV-J isolate) HRPO horse radish peroxidase IAHL International Avian Health Laboratory (University of Melbourne) kb kilo-base ML myeloid leukosis PBMC s peripheral blood monocytes PCR polymerase chain reaction RT reverse transcriptase SPF poultry specified pathogen free poultry SP Ratio sample to positive ratio S+/- shedding/non-shedding SPF CEF s chicken embryo fibroblasts from SPF poultry UOM University of Melbourne v

6 Contents Foreword...III Acknowledgments...IV Abbreviations... V List of figures and tables...viii Executive Summary...IX 1. Introduction Objectives Background Introduction The Avian Leukosis Viruses (ALV's) Recognition, origin and evolution of ALV-J Diseases, pathogenesis and effects on production Transmission and shedding of ALV infections Recent publications Virology and Serology Results Introduction Avian Leukosis Virus Antigen test kit Avian Leukosis Virus sub-group J Antibody test kit Viral prototypes Choosing cells for propagation of ALV-J Determining endogenous p27 levels in proposed cell lines PCR characterisation of uninfected cell lines Comparison of cells for sensitivity of ALV-J propagation using a viral tissue culture viral stock Sensitivity assay using a buffy-coat viral stock Comparing the sensitivity of C/O CEF s and DF-1 cells under different culture conditions Australian ALV-J isolates ALV-J infection status in various Australian flocks C/O CEF s and DF-1: ALV-E background Co-isolations of ALV-J and ALV-A Recommendations Antigenic characterisation Introduction The antigenic micro-neutralisation assay Antigenic variation is observed in Australian ALV-J isolates Experimental infection studies Introduction The viruses and chickens used The experimental design Sampling procedures Pathogenicity of Australian isolates The advantages and disadvantages of PCR detection in feathers vi

7 7. Molecular Biological detection of ALV-J Introduction PCR detection of ALV-J PCR positive and negative controls The primer pair H5 and H7b are specific for ALV-J The primer pair H5 and env-a are specific for ALV-A The isolation and propagation of ALV-J from field samples ALV-J PCR with DNA extracted from Tumours and Feathers Conclusions Molecular Biological analysis of ALV-J Introduction PCR amplification of ALV-J env region Cloning and sequencing of the env region Sequence analysis and phylogeny Sequence comparisons Phylogenetic analysis of Australian isolates Conclusions Development of practical methodologies for the detection of ALV-J Introduction A 96 well culture format of C/O CEF s for virus isolation A comparison of 24 well format vs 96 well format Comparison of 96 and 24 well formats in field samples What Samples are recommended? Implications and recommendations References Appendices Appendix A: Sample preparation and handling Appendix B: Precautions to avoid PCR contaminations Appendix C: Collection of blood for ALV Monitoring Appendix D: C/O CEF Micro-culture for monitoring of ALV s Appendix E: Interpretation and calculations for ALV-Ag and ALV-J Antibody test Appendix F: ALV-J PCR conditions and procedure Appendix G: Buffy-coat preparation Appendix H: Genomic DNA extraction from C/O CF s and Feathers Appendix I: Preparation of C/O CEF s Appendix J: DNA molecular weight markers Appendix K: The 24 well culture procedure vii

8 List of Figures and Tables Figures 3.1 ALV-J: Virion structure and major antigens. 4.1 No spurious or non-specific products with ALV-J primers. 4.2 No spurious or non-specific products with ALV-A primers. 4.3 PCR detection of the sensitivity assay. 4.4 PCR detection of sensitivity assay (take 2). 4.5 PCR detection of buffy-coat assay. 4.6 Antibody SP ratio variations. 4.7 C/O vs DF-1 background with ALV-Ag ELISA. 7.1 Schematic of PCR primers and products. 7.2 Specificity of the primers H5/H7b. 7.3 Specificity of the primers H5/Env-A. 7.4 ALV-J PCR on isolates from different tissues. 8.1 ALV-J envelope PCR. 8.2 Schematic of sequencing strategy. 8.3 Phylogenetic analysis of Australian isolates. Tables 4.1 ALV prototypes obtained for use as controls. 4.2 Comparison of background p27 levels in a umber of cell lines. 4.3 Determining the sensitivity of different cells to ALV-J infection. 4.4 Isolation of ALV-J from buffy-coat in C/O CEF s and DF-1 cells. 4.5 Comparison of different cells using Australian isolates. 4.6 Summary of all submissions. 4.7 Consolidated summary of ALV detection and isolation. 4.8 ALV-J infection status in various Australian flocks. 4.9 Prevalence of ALV-J and antibody in broiler breeder flocks Confirmation results for Australian samples (3.03) forwarded to the AFRC Compton, UK in November Virus micro-neutralisation assay for various ALV-J isolates. 6.1 Juvenile body weights of broilers infected with Australian strains of ALV-J. 6.2 Ratios of bursa weight to body weights. 6.3 Ratios of spleen weights to body weights. 6.4 Mortality pattern in experimentally infected birds at different ages. 6.5 Infection status of ALV-J infected birds at different ages. 7.1 Primer sequences and specificity. 8.1 Identity of sequenced clones. 8.2 Sequence comparisons: Australian isolates to HPRS-103 and ADOL-Hc Sensitivity for detection of ALV-J and ALV-A in C/O CEF culture in 96 well format (+/- DEAE) versus 24 well format. viii

9 Executive Summary Avian leukosis viruses (ALV s) are retroviruses for which subgroups A and B have long been recognised as causes of virus-induced tumour diseases ( big liver ) in layer hens. Recently, and particularly important in disease significance since 1995, ALV Subgroup-J has emerged from genetic recombination between ALV exogenous and endogenous (cellular) elements. Importation of contaminated great grandparent (GGP) and grandparent (GP) meat breeder lines has however enabled ALV-J to enter many countries. Vertical transmission via egg-semen-embryo is the major means of persistence of ALV-J infections and control requires the removal of ALV s from broiler genetic stocks. Retroviruses such as ALV-J are inherently predisposed to genetic mutation, and ALV-J isolates from various locations around the world now show a diversity of molecular-antigenic characteristics. The major overseas broiler breeding companies have recently made significant progress in reducing ALV-J infection in their elite flocks. The collective wisdom of industry and of these scientists suggests however, that it will be more difficult to control ALV-J, than it was to control subgroups A and B in earlier decades. These increased difficulties reflect two key biological features of ALV-J, which are (1) the range of antigenic variation that is being observed amongst J-strains, (2) the much enhanced efficiency of ALV-J for infection via contact transmission in broiler chickens. During the past decade ALV-J has emerged as a serious cause of mortality and suboptimal performance in commercial broiler breeders. Since its discovery in the United Kingdom in 1991, ALV-J has been diagnosed in many countries, including in Australia since Significant economic losses can be associated with ALV-J infection. The loss rate in commercial broiler breeders infected with ALV-J and intercurrent stressors can be as high as 1.5% per week in excess of normal mortality from approximately 20 weeks of age onwards. Consequential and replacement costs for these breeders can present a major economic loss for the poultry industry. The ALV-J associated loss rates, which have been reported in breeders worldwide, vary from 3-20%. Progeny broilers infected with ALV-J also tend to show reduced growth, unevenness of growth rates within flocks and a greater susceptibility to developing serious diseases when challenged by immunosuppressive viruses or secondary bacterial invaders. With this background knowledge of ALV-J and the clear potential of ALV-J to cause significant economic losses through mortalities and reduced productivity in Australia s chicken meat industry, the current project was undertaken during with the following experimental and operational objectives: Develop the virological and serological test systems required to enable culture and detection of the presence of ALV-J infection in vitro and to undertake comparison of isolates antigenically. Develop for Australian application the molecular biological techniques required for ALV-J identification and final diagnosis, with recognition of any genomic variation occurring amongst isolates. Develop practical methodologies for diagnosis of ALV-J related field problems in broiler flocks. Develop an active ALV-J international information network. Virological culture and serological investigations were undertaken which included comparative assessments of the effectiveness of commercially available avian leukosis ELISA-based reagents, as well as extensive investigations into the propagation of ALV-J in diploid chicken embryonic fibroblasts (CEF) cell cultures as well as a transformed CEF cell line (DF-1). The DF-1 line was imported as part of the present investigations and is classified as C/E (will propagate all sub-groups ix

10 of ALV except ALV-E). The most sensitive substrate for the detection of ALV-J has consistently been found to be C/O (chicken embryo fibroblasts which can propagate all ALV subgroups) cultures, which are prepared from a small SPF (specified pathogen free) flock, flock, which is maintained at the University of Melbourne for the purposes of this project. Standard SPF CEF cultures have been found to contain unacceptably high levels of endogenous leukosis viral antigens, while DF-1 cell cultures have been found to lack adequate sensitivity (50 to fold less) for isolation of ALV-J compared to C/O SPF cell cultures. Using the C/O SPF cell culture test systems for detection of ALV-J, samples were examined from the broiler breeder flocks of collaborating commercial organizations in Australia. In summary the following results were obtained during the project: (1) ALV-J infection of broiler breeders has been detected in grandparent (GP) and parent (P) flocks being maintained in Qld, NSW, Victoria and South Australia. (2) Detection of virus: ALV-J has been isolated in cell culture and identified by polymerase chain reaction (PCR) testing in field samples of tumours, whole blood, buffy-coat, albumens, meconium and also in feather follicles. The sample of choice which has been selected for screening flocks is to use buffy-coats from whole blood, while if large numbers of birds are to be tested, serums can be used most effectively. (3) Detection of antibody: The IDEXX commercial ELISA system for detection of antibody to ALV- J has been validated, with the reservation that low grade reactions and a prevalence rate of 15% or less are not indicative of flocks infected with ALV-J. Above these prevalences this ELISA test for antibody can be useful i.e. valid when used as a general flock test for detection of ALV-J infection. (4) While some commercial broiler breeder organisations tested in Australia showed apparent freedom from ALV-J in , examination of other organizations have shown that some 30% of the flocks (grandparent and parent) tested were positive. ALV-J virus was able to be isolated from most of these positive flocks. (5) In all, some 110 isolates of ALV-J (as well as 14 isolates of ALV-A) have been obtained during this study in Australia to date but the disease significance of ALV-A in these broiler stocks is not yet clear. However the presence of ALV-A could well be conducive to recombination with ALV-J, hence careful consideration needs to be given to removal of all exogenous (infectious) leukovirus by the elite breeder organisations in future. (6) Of further concern was the finding that ALV-A was frequently detected along with ALV-J. Mixtures of these viruses were obtained in some 16 of the isolates of ALV-J made from the field in Australia in The reasons for this very high rate of co-infection detected can only be speculative at present. (7) Experimental infection studies performed using broiler breeder stocks known to be free of ALV-J contamination using Australian ALV-J strains, UOM-201 and UOM-224, found both, when inoculated into day old chickens, to be capable of producing neoplasm, mortalities and eggtransmission of infection (see Chapter 6). Molecular biological techniques for ALV-J identification, final diagnosis and investigation of genomic variation in Australian isolates were developed. The results of studies undertaken (see Chapter 5 and 7 for details) have conclusively demonstrated that the molecular biological detection techniques i.e. Polymerase Chain Reaction (PCR) tests required for detection of ALV-J have been successfully established in this project. Primer sets H5/H7b were found to be able to demonstrate the presence of all Australian isolates of ALV-J in tumours, feathers, buffy-coats, albumens and meconiums. Additional primers (H5/env-A) were required to be developed for the discrimination of x

11 ALV-A from ALV-J. This was necessitated by the finding in these studies of a much higher prevalence of ALV-A infection than expected in Australian broiler breeder stocks. Sequencing examinations of the env portion of the genome of four ALV-J isolates from Australia, and their comparison with the UK (HPRS-103) and USA prototypes (ADOL-Hc1) strains of ALV-J, show that the Australian isolates are more closely related to one another than to the overseas strains. Hence the evolution of ALV-J would appear to be continuing in Australian breeder stocks. A preliminary observation to the effect that the genome sequences of Australian ALV-J strains would appear to be more analogous to those of the UK than the USA strains is still under investigation. Robust methodologies for the diagnosis of ALV-J involvement in field problems in broiler (breeder) stocks have also been developed in the course of this project. The recommended culture method using a 7-9 day passage period in C/O SPF cell cultures for buffy-coat or serum, followed by ELISA testing for ALV gs antigen p27 and final identification as ALV-J by PCR using primers sets will identify the presence of ALV-J in those birds showing disease but not tumours. While the ALV-J virus will be detected by this system in feather follicles or egg albumen, the diagnostic specimen of preference is whole blood. For breeders in which tumours are apparent eg. located at the sternum, liver or heart, the PCR test can be applied directly and rapidly (within 48 hrs) after extraction of DNA. In Australia, PCR differentiation of ALV-J from ALV-A should also be undertaken using PCR testing for final identification of isolates from field flocks. International reference networking was able to be established from early in this project with the two major international reference laboratories for ALV-J i.e. the Compton Institute of Animal Health, UK (Dr. Venugopal Nair) and the Avian Diseases and Oncology Laboratory (ADOL) of the USDA (Dr. Ally Fadly). Positive relations with exchange of information and feedback have also been fostered with two of the major breeding companies in the world which supply breeding stock into Australia i.e. Aviagen (Scotland) and Cobb Vantress (USA). xi

12 1. Introduction In recent years, avian leukosis virus subgroup J (ALV-J) infections in breeders and broilers have become recognised as causing significant economic problems world-wide. While the major overseas broiler breeding companies based in the USA and the UK have made significant progress in the reducing incidence of infection in their elite stocks for export concerns still exist that ALV-J may be circulating in the Australian industry RIRDC supported a scoping project of 12 months duration, conducted by the University of Melbourne, which was completed in June Serological evidence was reported showing the occurrence of ALV-J infections within broiler breeder flocks in New South Wales, Queensland and Victoria. Previously considered exotic to Australia, this vertically and horizontally transmitted viral pathogen appeared to be present to some extent in Australian flocks. Overseas experiences with ALV-J disease, especially during 1997 and 1998, saw heavy losses incurred in breeding flocks in Europe and the USA (Van der Sluiss, 1998). Capable of causing tumour diseases in breeders and suboptimal growth and liveability in broiler flocks, ALV-J is currently a major potential cause of economic losses across the Australian chicken meat industry. Research has indicated that the difficulties in eradicating ALV-J, compared to ALV subgroups A and B in earlier decades, arise primarily because of two biological features. These are the range of antigenic variation being observed amongst strains, and the enhanced efficiency of ALV-J for contact transmission and spread amongst young chickens. Overseas, the major primary breeding companies are endeavouring to eradicate ALV-J from their elite stocks. Hopefully these programs will eventually be successful. However, a strong case exists for developing an Australian capacity to be able to competently detect ALV-J infection. 2. Objectives Develop the virological and serological test systems required to enable culture and detection of the presence of ALV-J infection in vitro and to undertake comparison of isolates antigenically. Develop for Australian application the molecular biological techniques required for ALV-J identification and final diagnosis, with recognition of any genomic variation occurring amongst isolates. Develop practical methodologies for diagnosis of ALV-J related field problems in broiler flocks. Develop an active ALV-J international information network. 3. Background 3.1 Introduction There is considerable background knowledge of avian leukoviruses (ALV's) that is useful to form an understanding, both of the current ALV-J disease situation and of the technologies that are needed to work with ALV-J. In the last decade, there have been numerous investigations undertaken using molecular biological techniques to better understand the basis of ALV-J antigenicity and pathogenicity, as well as improving detection technologies. Some relatively recent reviews of ALV's and ALV-J are the Special Supplement to Avian Pathology (1998), Fadly and Winter (1998), Spencer (1999) and Fadly and Payne (2003). 1

13 The presence of ALV-J in Australian broiler breeders and its likely transmission to commercial broiler flocks was detected during an RIRDC scoping study undertaken to investigate causes of suboptimal broiler productivity in Australia during (Bagust et al., 1999). However, a full virological investigation has not been undertaken. The research task of providing Australia's broiler industry with the tools i.e. laboratory technologies and reagents, that it will need to effectively control endemic ALV-J disease, is a significant undertaking for industry, scientifically as well as economically. 3.2 The avian leukosis viruses (ALV's) ALV's are the major grouping within the avian oncornaviruses (tumour-causing RNA viruses). The minor known avian oncornaviruses include reticuloendotheliosis virus (REV) (Bagust, 1993) and lymphoproliferative disease of turkeys (Biggs, 1997). These latter viruses however tend to be natural infections of non-chicken avian species, producing only sporadic and opportunistic infections in chickens. Figure 3.1 ALV-J: Virion structure and major antigens. Avian Leukosis Virus sub-group J (ALV-J): gp 37 TM CA p27 NC p12 gp 85 SU RT p68 IN p32 PR p15 gag pol env ALV-J (7841 bp) ALV s that naturally infect chickens are divided into six subgroups, being designated A, B, C, D, E and J. Each subgroup can be differentiated by their host range and viral envelope antigens (gp85). The env gp85 of ALV-J has only 40% overall homology to the corresponding sequences of A-E (Bai et al., 1995 a, b). ALV-Ag ELISA is useful for the detection of the p27 antigen which is common to all sub-groups of ALV. However, the specific identification of ALV-J requires antigenic methods or a molecular biological approach such as PCR. 2

14 Of the six subgroups known to occur in chickens, five are exogenous i.e. the complete virus will be expressed from cells, is infectious and potentially neoplastic. Subgroups A and B have classically been those of field significance in leukosis tumours, especially A. Subgroup E ALV's however are primarily endogenous i.e. the viral nucleic acid is permanently integrated into the cellular genome, replicates with the cell and is transmitted with it (genetically). Endogenous ALV's are rarely expressed in complete infectious form and are of extremely low pathogenicity. However these may produce low levels of ALV group-specific (gs) antigen p27, producing background reactions which can cause difficulties in detection of other ALV subgroups e.g. by ELISA testing for gs antigen. Other subgroups of ALV s that have been isolated were from pheasants (subgroups F and G), Hungarian partridge (H) and Japanese quail (subgroup I). Hence when discovered (Payne et al., 1991) the designation of subgroup J was given to this most recently isolated ALV subgroup. ALV-J in the field has occurred almost entirely in meat type chickens. Layers have rarely become infected (Payne, 1999 pers. comm; Spencer, 1999 pers. comm.) but experimental studies with Rous Sarcoma virus (ALV-J) envelope pseudotypes made by Payne et al. (1992) have shown that both layers and turkeys are susceptible to ALV-J infection, while other poultry and game birds appear resistant. In chickens, layer strains should however, be noted to be potentially at risk in Australia and their exposure to ALV-J contamination should be avoided. The genome of oncornaviruses is single-stranded RNA, for which integration of a provirus into the cellular genome occurs for exogenous ALV's during replication, or permanently in the case of endogenous ALV's (subgroup E). Replication of oncornaviruses requires the production of a DNA intermediate from the viral RNA template, using the viral enzyme reverse transcriptase (RT) encoded by the pol gene region in the viral genome. Production of the avian leukosis group specific antigen (p27) is also common to ALV subgroups A-J and is located in the gag gene region. The proviral genetic organisation of HPRS-103 is LTR (long terminal repeat region) - leader - gag/pol - env - LTR in common with other ALV subgroups, whilst no viral oncogene sequence (myc) could be detected for HPRS-103 (Bai et al., 1995 a, b). 3.3 Recognition, origin and evolution of ALV-J ALV-induced leukotic diseases have not been a problem in meat strains, most of which were considered to be resistant to infection by the common ALV subgroups. In 1988 however, during a study undertaken by Payne of the ALV status of broiler breeders in the UK, several isolations of ALV were made from clinically normal breeders, and also from a case of myeloid leukosis (myelocytomatosis) (ML). Study of these viruses resulted in characterisation of the new J subgroup for which one isolate, HPRS-103, was designated as the prototype (Payne et al., 1991). Since 1996 however, the poultry meat industry world-wide has been economically damaged by serious losses in broiler breeders. Also around this time, broiler breeder flocks experiencing relatively high rates of ML also became apparent in the USA, and subgroup J-like isolates were detected (Fadly and Smith, 1997). Clinical reports of the disease were occurring from around the world during 1997 and 1998, (Van der Sluiss, 1998) and " The relatively sudden global appearance of the disease seemed to reflect a widespread dissemination of a vertically transmitted virus from infected primary breeding stock " (Payne 1999 pers. comm.) Clues to the origin of ALV-J come from the finding (Bai et al., 1995 a, b) that the env gene of HPRS- 103 has 75% homology with the EAV-O family of endogenous avian retroviruses, suggesting that ALV-J arose as a result of genetic recombination between an exogenous ALV and env J sequences, likely followed by further mutation (Payne, 1998). Comparisons of sequence data between HPRS- 103 and two USA isolates also indicate they arose from a common ancestor (Benson et al., 1998). These findings, as well as the timing of the discovery of ALV-J, would therefore suggest that ALV-J arose as a single occurrence some years ago, then has undergone mutations subsequently. Like the env gene of other retroviruses (such as HIV), that of ALV-J shows a tendency to mutate whilst like many other RNA viruses (such as V), absence of a proof-reading mechanism for ALV- 3

15 J means that errors in RNA replication cannot be corrected. When the env gene sequence in 12 new isolates of ALV-J were studied by Venugopal et al. (1998), all were found to differ from one another, and from HPRS-103, showing amino acid sequence identity of %. Furthermore, most of the recent isolates failed to be neutralised by antisera to HPRS-103. It would therefore seem most likely that immune responses are exerting the selection pressure which drives this antigenic variation. Such variation has also been observed in USA isolates, in that some of their most recent isolates are not being neutralised by antibody to their original prototype strain ADOL-Hc1 (Fadly, 2000 pers. comm.). Whilst antibody to the USA's prototype ALV-J, ADOL-Hc1, can neutralise the UK's HPRS-103, antibody to HPRS-103 did not reciprocally neutralise ADOL-Hc1. Hence even these two prototype strains must be considered antigenically related, but not identical (Fadly and Smith, 1999). 3.4 Diseases, pathogenesis and effects on production Lymphoid leukosis, formerly called big liver, is the most commonly naturally occurring tumour caused by ALV's, particularly those of subgroup A and, to a lesser extent, B. A further wide range of ALV tumours which have been reported world-wide, include nephroblastomas, haemangiomas, osteopetrosis and myeloblastosis-myelocytomatosis. Major determinants of the type of tumour produced include the host genotype, the strains of virus and the exposure dose of ALV. Lymphoid leukosis (LL) tumours are predominantly B-cell lymphomas, reflecting their origin in the bursa of fabricius of the young chicken. ALV-transformed bursal lymphocytes then metastasise to the liver and other visceral organs. As the incubation period is rarely less than 14 weeks, LL is usually a tumour disease of older chickens, i.e. commercial egg-layers. ALV's are known to be able to multiply in virtually all tissues and organs of susceptible chickens but the richest concentrations occur in the medullary macrophages in the bursa, the sheathed capillaries in the spleen and throughout the myocardium of the heart. Sites in the host from which ALV's may be released into the environment include the magnum of the oviduct and the Lieberkuhn glands in the intestine. This point can be seen as critical, as high levels of ALV's may be shed as infectious virus or antigen into the albumen of the egg, and thence into the allantois of the chicken embryo (see 3.5 Transmission and Shedding). While ALV s of subgroup A have long been recognised to cause sporadic mortalities from lymphoid leukosis tumours, the greatest production losses were more recently described in landmark papers by Canadian scientists (Gavora et al., 1980; Gavora et al., 1982), and included reductions of eggs per hen housed for a single lay cycle. Negative effects included egg weight and shell thickness as well as fertility, chick hatchability, rate of growth and liveability. Following these overseas findings, both the chicken meat and egg industry research funding bodies established research projects for the development of ALV detection technology (e.g. Ignjatovic and Bagust, 1982) which subsequently enabled successful leukosis reduction/eradication strategies for subgroups A and B to be undertaken in Australia. ALV-J infection in broiler breeder flocks is associated with the occurrence of myeloid leukosis (myelocytomatosis) or ML. ML is a tumour condition which is readily characterised as comprised of transformed white blood cells from the bone marrow. First observed in broiler breeder birds between weeks of age. (Payne, 1991), ML tumours are now being reported to appear in the field as early as 17 weeks (Zavala, 1998). Zavala notes that the timing, however, may vary according to "factors such as genetics, environment, management, nutritional status, concomitant infections, immunocompetence and (the) actual form of transmission" (i.e. congenital or horizontal - see next Section) Field experience indicates that immunosuppressive infections such as infectious bursal disease, chicken anaemia or Mareks disease viruses, i.e. immunosuppressive conditions. "are a lethal combination with ALV-J infection" (Zavala, 1998). Further, Payne (1998) has now mooted the likelihood of ALV-J strains occurring which will operate in field flocks as acutely transforming leukotic viruses, i.e. having acquired oncogenes. Both these authors report that ML mortality rates may reach 6-8% per month, and will devastate the hen-housed egg production of broiler breeder 4

16 flocks. This has also been confirmed in Australia (G. Richards 1998, pers. comm.), while the observed lack of uniformity of broiler progeny and uneven egg size have also been conjectured as caused by ALV-J (P. Scott 1999, pers. comm.). The frequency of tumour types seen by Payne 1998 (and pers. comm. 1999) during from suspect ALV-J infected flocks in Europe were ML (58%), histiocytic sarcoma (12%), erythroblastosis (9%) and blast cell tumours (5%). Numerous birds submitted had more than one type of tumour, and some up to four different types. In experimental cases of ML, Payne found enlargement of the liver to occur in 86% of cases followed by "skeletal myelocytomas (56%), particularly on the inner surface of the sternum". Zavala (1998) confirms and extends this latter finding (which may well be pathognomic for ALV-J?) of myelocytomas "around the skull, in the mucosa of the larynx and trachea. and around the ribcages and keelbone". Such lesions are now being observed at autopsy of Australian broiler-breeders (Harrigan 1999, pers. comm.). While the HPRS-103 strain of ALV can replicate in a wide variety of tissues including the adrenals, heart, proventriculus and other parts of the gastrointestinal tract (Arshad et al., 1999), HPRS-103 has also been found to exhibit a high tropism for cultured monocytes - but a low tropism for bursal follicles, i.e. the reverse to subgroup A of ALV. Such tropism may well explain why ALV-J induces ML rather than LL, and also the ability of ALV-J to also produce lesions in distinctly different sites to LL, e.g. the costro-chondral junctions. Until very recently, the published scientific documentation was not sufficient to enable a clear understanding of ML effects on broiler health (Payne, 1998; Zavala, 1998), although the effects of ML have been conjectured as significant by numerous industry scientists, e.g. Goodwin (1999). The journal, Avian Diseases 1999 however, includes a report that the body weights of ALV-J positive broilers, monitored between 1-8 weeks of age, were only some 64% of those of ALV-J negative broilers (Stedman and Brown, 1999). No co-infection by another avian pathogen was detected in either group. Concurrently, reports have come from the USA of the isolation of ALV-J from parent meat-type chickens experiencing ML as early as 6 weeks of age, and ALV-J being obtained from commercial broilers at 4 weeks of age (Fadly and Smith, 1999). Canadian studies on ML-infected breeders have also shown that the small eggs were more likely to be infected with ALV-J than large eggs (Spencer et al., 1999) so effects on egg size can also be imputed for ALV-J. 3.5 Transmission and shedding of ALV infections Exogenous subgroups of ALV s (A-D and J) show two main transmission mechanisms: Congenital. Transmission is mediated by virus shedding to egg albumen and infection of the embryo. Most of these chickens will become immunologically tolerant viraemics (V+) without antibody (A-) and become shedders (S+) of gs antigen and virus. Both can be detected in cloacal or vaginal swabs and egg albumens using gs ELISA tests. V+A-S+ hens will congenitally transmit ALV s to their progeny which persistently shed throughout their lives. Furthermore, the large amounts of infectious ALV in their meconium (up to 100 infectious units per gram) make these shedding chickens a serious danger to their uninfected hatchmates. Horizontal. Infection occurs through close contact with hatchmates or penmates. Chickens which are first exposed to ALV s after hatching become, depending on their age and hence susceptibility to infection, either V+A-S+ or antibody positive birds. Most of the second type are non-shedders (V- A+S-) but some may become continual shedders (V-A+S+). Stress and other intercurrent infections are known to enhance tumour formation and most importantly to also increase the frequency of shedding by ALV-infected breeder hens. Specifically for ALV-J, Zavala (1998) notes a daunting list of the management husbandry stressors that can contribute to these aspects. These include high bird density, deficiencies in feeder or drinker space, nutritional imbalance, male-female ratios being inadequate and vaccine overload, in addition to the extreme importance of controlling or preventing immunosuppressive diseases. 5

17 Earlier experiences with ALV eradication programs involved White Leghorn (layer) strain chickens and exogenous ALV subgroups A and B. Results indicated that young hatched chickens could be expected to develop an age resistance to ALV horizontal infection within the first week, or even by several days of age. Following exposure to ALV's such chickens became immune, i.e. status V-A+Sand did not continue to shed virus into their environment or their eggs/embryos. For ALV-J transmission the problems appear far more complicated however in that meat-type birds appear to be particularly prone to developing tolerant viraemic (life-long) infections following exposure post-hatching and the period of susceptibility to developing this V+A-S+ status is believed to be as long as 6 weeks (Payne 1998). 3.6 Recent publications Previous publications have reported the adverse effects and pathogenicity of subgroup J avian leukosis virus (ALV-J). Further evidence for ALV-J s negative impact on body weight uniformity and liveability in breeder flocks (Zavala, 1998), egg weight and shell quality (Spencer et al., 2000), and progeny performance and liveability (Goodwin et al., 1999; Stedman et al., 1999; Zavala, 1998) have been further explored. Methods of viral transmission have also been researched to aid in the eradication of ALV-J from broiler breeder flocks (Witter et al., 2000; Witter et al., 2001; Koch et al., 2000). Profiles of infection with ALV-J and factors that predict virus transmission to progeny have been studied in detail by Witter et al. (2000). The results obtained largely validate the screening procedures currently being used in Australia to identify potential transmitter hens. It is concluded that in infected flocks, detection of all transmitter hens by such screening procedures is unlikely. Thus, eradication programs which are based solely on dam testing may be less effective than those where dam testing is combined with procedures to mitigate early horizontal transmission in progeny chicks. Horizontal transmission of ALV-J can be reduced in broiler breeder stocks when these are hatched and reared in small groups (Witter et al., 2001). Serological profiling of ALV-J infected chickens has also been carried out (Hwang et al., 2002). The results indicated that the gs antigen of ALV-J-infected flocks increased, but that of the uninfected flocks decreased during young ages. The anti-alv-j antibody of infected flocks was higher and increased earlier than that of uninfected flocks. Thus, measuring gs antigen in blood at the ages of 1 and 6 weeks by ELISA is suitable to discriminate between ALV-J-infected flocks and uninfected flocks having ALV-E i.e. endogenous non-tumorigenic avian leukosis activity. Data has also been published that demonstrates that the ALV-J status of caged males has no influence on sperm quality or hatchability (Benton et al., 2002). It should be noted however that ~40% of males in this study died by 43 wks of age due to the effects of ALV-J. ALV-J continues to undergo antigenic variation and recombination (Chesters et al., 2001; Fadly et al., 2000; Gingerich et al., 2002; Lupiani et al., 2000; Silva et al., 2000; Venugopal et al., 2000) and has been isolated in a number of regions around the world (Du et al., 2000; Du et al., 2002; Jurajda et al., 2000). Several studies have expanded upon the knowledge of how and when to detect ALV-J by PCR and in situ hybridisation (Stedman et al., 2000; Sung et al., 2002; Zavala, et al., 2002). In Australia the application of molecular techniques for the detection and characterisation of ALV-J have been developed and established (Bagust et al., 2002; Fenton et al., See Section D). 6

18 4. Virology and Serology Results 4.1 Introduction The main objectives of this project were to introduce and develop technologies for the propagation and detection of ALV-J for the Australian poultry industry. Initially propagation of various controls, including the sub-groups of ALV s, was required to establish the procedures and techniques needed for detection of ALV-J. ALV-Ag ELISA is useful for the detection of the p27 antigen which is common to all sub-groups of ALV. However, the specific identification of ALV-J requires antigenic methods or a molecular biological approach such as PCR. The procurement of the necessary controls and the characterisation of proposed cell lines for propagation of ALV-J was therefore the essential first step in the establishment of such procedures. This chapter describes the process undertaken to establish the serological and virological procedures required for investigation of ALV-J. 4.2 Avian leukosis virus antigen test kit FlockChek ALV-Ag is an enzyme immunoassay from IDEXX laboratories (product code ) for the detection of avian leukosis virus antigen p27. The p27 antigen is common to all sub-groups of ALV including endogenous viruses. Thus the ALV-Ag ELISA is only useful to determine the presence or absence of ALV s in a particular sample. It cannot be used to distinguish between the individual sub-groups of the ALV s. Positive samples identified by ALV-Ag ELISA need to be further investigated to determine their exact sub-group, be it A, B C, D, E or J. This can be achieved using the polymerase chain reaction (PCR, which is described in Chapter 7). The recommended sample types for the IDEXX ELISA kit are light albumen or cloacal swab samples. While serum has been validated for use on the ALV-Ag test, it is not a recommended sample for the detection of exogenous virus because of potential interference from endogenous sequences. Testing of tissue culture medium after virus isolation is possible with this kit, however it should be noted that certain cells used for virus propagation may also contain high levels of background p27 expression and their ability to support propagation of ALV-E should be investigated prior to use as a detection system for ALV s. The ALV-Ag IDEXX kit is in a micro-titration format in which anti-p27 antibody is coated onto 96-well plates. Sample p27 forms a complex with the coated antibody. After washing away unbound material, an anti-p27: horseradish peroxidase (HPRO) conjugate is added which binds to attached p27. In the final step of the assay, unbound conjugate is washed away and enzyme substrate is added to the well. Colour development may then be related to the amount of p27 present in the test sample. 4.3 Avian leukosis virus sub-group J antibody test kit FlockChek ALV-J Antibody test kit from IDEXX laboratories (product code ) is an enzyme linked immunosorbent assay for the detection of antibody to ALV-J in chicken serum. The ALV-J Antibody test kit detects antibody produced, usually following horizontal transmission of the ALV-J virus. The assay has been developed in the microtiter format where by ALV-J gp85 antigen has been coated onto 96-well plates. During incubation of the test sample in the coated well, antibody specific to ALV-J gp85 forms a complex with the coated antigen. After washing unbound materials away from the wells, a (Goat) anti-chicken immunoglobulin: horseradish peroxidase (HRPO) conjugate is added that binds to any attached chicken antibodies in the wells. In the final step of the assay, unbound conjugate is washed away and an enzyme substrate, hydrogen peroxide, and a chromogen are added to the wells. Subsequent colour development may then be related to the amount of anti- ALV-J present in the test sample. The ALV-J Antibody test kit has been developed as a flock screening tool for monitoring horizontal transmission of the virus. ALV-J seroconversion is variable across lines and may depend on endogenous leukosis virus expression (Smith et al., 1990). Testing of meat-type birds less than weeks of age is not recommended. A positive result on the ALV-J antibody test kit indicates exposure to the ALV-J virus; antibody titre does not indicate whether the virus is being actively shed. Hence, determination of ALV-J flock status should include testing for 7

19 the virus. Vertical transmission of ALV-J through the eggs of infected breeders will usually result in seronegative immune tolerant progeny, which subsequently then transmit ALV-J as adult hens and roosters. 4.4 Viral prototypes A number of ALV prototypes have been obtained for use by the International Avian Health Laboratory (IAHL) at the University of Melbourne. These control viral stocks are for use as controls to establish the conditions necessary for successful propagation and specific detection of ALV-J. These control stocks are summarised in Table 4.1. Table 4.1 ALV prototypes obtained for use as controls. Virus Subgroup Use/Comments RAV-1 ALV-A RSV-A ALV-A RAV-2 ALV-B RAV-49 ALV-C RAV-50 ALV-D RAV- ALV-E ADOL-Hc1 ALV-J ADOL-7501 ALV-J HPRS-103 ALV-J J98290/191 ALV-J Proviral DNA extracted and used for a PCR control. Viral stock used routinely as a tissue culture positive for viral isolation Proviral DNA extract and used for a PCR control Proviral DNA extract and used for a PCR control Proviral DNA extract used for PCR control Proviral DNA extract used for PCR control USA prototype ALV-J isolate. Proviral DNA extract used for PCR control A USA isolate which is a transforming/oncogenic virus. Proviral DNA extract used for PCR control The original ALV-J prototype from the UK. Proviral DNA extract used for PCR control The first Australian isolate to be identified. Isolated in 1998 by Gordon Firth of Intervet. Proviral DNA extract used for PCR control. Viral stock used occasionally as a tissue culture positive for viral isolation Note: ALV-J strains HPRS-103 (prototype, UK) and ADOL-Hc1 (prototype USA) were obtained from Dr. G Zavala, University of Georgia, USA. The Australian reference strain of ALV-J, J-98290/191, was provided by Dr. G.Firth, Intervet Australia. 4.5 Choosing cells for propagation of ALV-J A number of different cell types and cell lines are available which are suitable for the propagation of ALV-J. Each of these has different properties that need to be considered when using these cells for virus isolation. The cell lines include C/O CEF s (which will propagate all sub-groups of ALV s), DF-1 cells (ATCC CRL a transformed CEF cell line that is C/E). SPF chicken embryos from SPAFAS Australia were also examined for use as a source of CEF s for the propagation of ALV s. The characterisation of each of these cell types was undertaken in order to determine their suitability for the isolation and detection of ALV-J. 4.6 Determining endogenous p27 levels in proposed cell lines 8

20 Initially it is important to establish the background or endogenous levels of ALV-Ag in any cells proposed for use in virus isolation and propagation. Three cells lines, C/O CEF s, DF-1 s and SPAFAS CEF s, were cultured under normal conditions for four sequential passages and the level of p27 antigen measured by ALV-Ag ELISA. Table 4.2 shows the average SP ratio (Appendix E) recorded in each of these cell lines. SPAFAS CEF s showed an average background SP ratio of 0.32 (>0.20 is considered positive). This relatively high SP ratio is due to the endogenous expression of the p27 protein in these cells. This background level makes these cells unsuitable for use in virus isolation as the degree of sensitivity required for examining field samples which may contain low levels of virus will be lost in this background. C/O CEF s have a much lower background SP ratio of 0.06, while DF-1 cells showed an average SP ratio of Thus both C/O CEF s and DF-1 cells were found suitable for the isolation and propagation of ALV s based on their low background expression of the ALV p27 protein. Table 4.2 Comparison of background p27 levels in a number of cell lines. Cell OD reading SP ratio Mean C/O CEF s P C/O CEF s P C/O CEF s P C/O CEF s P DF-1 cells P DF-1 cells P DF-1 cells P DF-1 cells P SPF CEF s P SPF CEF s P SPF CEF s P SPF CEF s P Kit Positive control SP Mean 0.06 SP Mean 0.01 SP Mean 0.32 Kit Positive control SP Mean 1.00 Kit Negative control Kit Negative control SP Mean PCR characterisation of uninfected cell lines It is important to establish the background or endogenous levels of product which may be spuriously or non-specifically amplified from any cells proposed for use in virus isolation and propagation of ALV s. Three cells lines C/O CEF s, DF-1 s and SPAFAS CEF s, were cultured under normal conditions for four sequential passages and the genomic DNA was extracted at each passage and tested by PCR with ALV-J and ALV-A specific primers (Chapter 7). Each of these cell lines was also infected with ALV-J and ALV-A as positive controls for viral infection and growth under these cell culture conditions (Appendix K). In order for these cell lines to be useful for virus isolation and detection of ALV s by PCR after culture, no observable background should be detected in uninfected cells. Figure 4.1 demonstrates the absence of any detectable PCR products in any of these uninfected cell lines. It also demonstrates that all three cell lines can propagate ALV-J by the PCR amplification 9

21 of the expected product (544 bp) when using the primers H5/H7b (Chapter 7). Figure 4.2 demonstrates that each cell line can also propagate ALV-A by the PCR amplification of the expected product (694 bp) when using specific primers H5/Env-A (Chapter 7) bp No Amplification in uninfected cells Amplification of control ALV-J Figure 4.1 No spurious or non-specific products with ALV-J primers. Three cell lines, C/O CEF s, DF-1 and SPAFAS CEF s, were cultured for four sequential passages and genomic DNA extracted after each passage and subjected to PCR with the primers H5/H7b (Chapter 7). Each cell line shows the absence of any amplified products at each of the four passages (lanes 2-13). These cell lines were also infected with ALV-J and the presence of the expected 544 bp product from each of the infected cell lines by H5/H7b PCR indicates each of these cell lines is able to support the propagation of ALV-J (lanes 15-17). 1. DNA molecular weight markers (Appendix J) 2. C/O CEF s passage 1 (H5/H7b). 3. C/O CEF s passage 2 (H5/H7b). 4. C/O CEF s passage 3 (H5/H7b). 5. C/O CEF s passage 4 (H5/H7b). 6. DF-1 passage 1 (H5/H7b). 7. DF-1 passage 2 (H5/H7b). 8. DF-1 passage 3 (H5/H7b). 9. DF-1 passage 4(H5/H7b). 10. SPAFAS passage 1(H5/H7b). 11. SPAFAS passage 2 (H5/H7b). 12. SPAFAS passage 3 (H5/H7b). 13. SPAFAS passage 4 (H5/H7b). 14 DNA molecular weight markers. 15. ALV-J infected C/O CEF s (H5/H7b). 16. ALV-J infected DF-1 cells (H5/H7b). 17. ALV-J infected SPAFAS CEF s (H5/H7b). 18.ALV-J PCR positive control (H5/H7b). 19. PCR negative control (H5/H7b). 10

22 bp No amplification in uninfected cells Amplification of control ALV-A Figure 4.2 No spurious or non-specific products with ALV-A primers. The three cell lines, C/O CEF s, DF-1 and SPAFAS CEF s, were infected with ALV-A. The presence of the expected 694 bp product from each of these infected cell lines after PCR when using the primers H5/Env-A (Chapter 7) demonstrates the suitability of each of these cell lines for the propagation of ALV-A (lanes 5-7). The absence of any product in the 4th passage from any of these cell lines with ALV-A primers demonstrates the absence of any non-specific or spurious amplification in all these cell lines (lanes 2-4). 1. DNA molecular weight markers. 2. C/O CEF s passage 4 (H5/Env-A). 3. DF-1 cells passage 4 (H5/Env-A). 4. SPAFAS CEF s passage 4 (H5/Env- A). 5. ALV-A infected C/O CEF s (H5/Env-A). 6. ALV-A infected DF-1 cells (H5/Env-A). 7. ALV- A infected SPAFAS CEF s (H5/Env-A). 8. ALV-A isolate propagated in C/O CEF s (H5/Env-A). 9. ALV-A positive control (H5/Env-A). 10. PCR negative (H5/Env-A). 4.8 Comparison of cells for sensitivity of ALV-J propagation using a viral tissue culture viral stock For virus isolation the sensitivity of the cell line to infection by the virus is a critical factor to consider. A cell line that requires the presence of a high level of virus for infection and propagation to occur is disadvantageous i.e. lacks sensitivity when isolating virus from samples and tissues with potentially very low levels of viable virus. The three cell lines C/O CEF s, DF-1 cells and SPAFAS CEF s were thus tested for their sensitivity to infection using a serially diluted stock of ALV-J. An ALV-J viral stock was serially diluted in 10-fold steps from 10-1 to 10-8 and each cell line was infected with these dilutions. The infected cells were then passaged for 2 x 5 days according to the standard protocol (Appendix K) and assayed by ALV-Ag ELISA. The genomic DNA from each serial dilution for each cell line was also extracted and subject to PCR with the H5/H7b primer pair. The results are summarised in Table 4.3. SPAFAS CEF s as in the initial uninfected characterisation showed a high background level by ALV-Ag ELISA (control uninfected cells SP-0.50), again demonstrating the unsuitability of these cells for virus isolation as it is not possible to accurately determine the point at which viral propagation ceases. DF-1 cells recorded a positive SP ratio demonstrating viral infection up to the 10-4 dilution, while the direct comparison to C/O CEF s recorded a positive SP ratio up to 10-7, indicating C/O CEF s have significantly increased sensitivity for ALV-J infection. This increased sensitivity was confirmed by the PCR detection of viral growth after extraction of genomic DNA from each dilution point. The results for the PCR analysis are shown in Figure 4.3. C/O CEF s were positive by PCR out to 10-5 whereas DF-1 infection was able to be detected only at 10-1 dilution. Both the ALV-Ag and PCR results demonstrate that C/O CEF s have a superior level of sensitivity for infection by ALV-J. In the order of x1000 (ALV-Ag detection) or x10,000 (PCR) fold 11

23 less virus was required to infect C/O CEF s compared to DF-1 cells. Given the important implications of this finding the experiment was repeated and the PCR results for the second experiment are shown in Figure 4.4. This experiment again showed an increased sensitivity of infection for C/O CEF s (x1000 fold at the PCR level), over the two cell types tested. It should be noted that the viral stock used for both these experiments had been passaged a number of times in C/O CEF s and it is possible that the virus may have undergone some adaptation or selection during these passages to become more infective in C/O CEF s than DF-1 cells. To test for this possibility, inoculations of ALV-J in cells obtained directly from an experimentally infected bird was undertaken (Section 4.9) Table 4.3 Determining the sensitivity of different cells to ALV-J infection. SP Ratio Dilution DF-1 C/O SPF Control Note: positive SP ratios are bolded and control represents uninfected cells. SPF CEF s C/O CEF s DF Figure 4.3 PCR detection of the sensitivity assay. Serial dilutions of a viral stock were inoculated into SPAFAS CEF's, C/O CEF's and DF-1 cells. Genomic DNA was extracted (Appendix H) from each dilution point and subjected to H5/H7b PCR. The limit of detection for each cell line was determined to be 10-3 for SPAFAS CEF s, 10-5 for C/O CEF s and 10-1 for DF-1 cells. 1. DNA molecular weight markers (Appendix J). 2. ALV-J positive control (H5/H7b) SPAFAS CEF s (H5/H7b) SPAFAS CEF s (H5/H7b) SPAFAS CEF s (H5/H7b) SPAFAS CEF s (H5/H7b) SPAFAS CEF s (H5/H7b) SPAFAS CEF s (H5/H7b) SPAFAS CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b). 12

24 C/O CEF s SPF CEF s DF Figure 4.4 PCR detection of sensitivity assay (take 2). The experiment from Figure 4.3 was repeated to confirm the increased sensitivity of C/O CEF s to ALV-J infection. The limits of detection were 10-6 for C/O CEF s, 10-4 for SPAFAS CEF s and 10-3 for DF-1 cells. Demonstrating a x100 fold increased sensitivity of C/O CEF s compared to DF-1 cells. 1. DNA molecular weight markers (Appendix J) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b). 10. Uninfected C/O CEF s (H5/H7b) SPAFAS CEF s (H5/H7b) SPAFAS CEF s (H5/H7b) SPAFAS CEF s (H5/H7b) SPAFAS CEF s (H5/H7b) SPAFAS CEF s (H5/H7b) SPAFAS CEF s (H5/H7b) SPAFAS CEF s (H5/H7b) SPAFAS CEF s (H5/H7b). 19 Uninfected SPAFAS CEF s (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b). 28. Uninfected DF-1 cells (H5/H7b). 29. PCR negative (H5/H7b). 30. DNA molecular weight markers (Appendix J). 4.9 Sensitivity assay using a buffy-coat viral stock In order to eliminate the possibility of viral culture adaptation leading to the greater sensitivity of C/O CEF s for virus isolation, blood from an experimentally infected bird was used to prepare a buffy-coat stock (Appendix G). This preparation was serially diluted and used as an inoculum for a similar experiment comparing the infectivity of C/O CEF s versus DF-1 cells in a 24 well format. Using a viral inoculum that had been passaged in vivo should overcome the problem of virus adaptation in culture. This inoculum should also closely reflect the expected viral load found in a field whole blood sample. Table 4.4 and Figure 4.5 again demonstrate the increased sensitivity of C/O CEF s for virus isolation even under these conditions. C/O CEF s showed a x50 fold higher sensitivity than DF-1 cells when using buffy-coat as an inoculum. 13

25 Table 4.4 Isolation of ALV-J from buffy-coat in C/O CEF s and DF-1 cells. Dilution SP ratio C/O CEF s DF-1 cells Control Note: positive SP ratio s are bolded and control represents uninfected cells. DF-1 C/O CEF s Figure 4.5 PCR detection of buffy-coat sensitivity assay. The sensitivity assay was repeated using serial dilutions of a buffy-coat preparation from whole blood obtained from a bird experimentally infected with ALV-J. Both C/O CEF s and DF-1 were infected and H5/H7b PCR used for ALV-J detection The limit of detection for DF-1 cells was and for C/O CEF's. 1. DNA molecular weight markers (Appendix J). 2. ALV-J positive PCR control DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b) DF-1 cells (H5/H7b). 10. Uninfected DF-1 cells (H5/H7b). 11. PCR negative control (H5/H7b). 12. ALV-J positive PCR control C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b) C/O CEF s (H5/H7b). 20. Uninfected C/O CEF s (H5/H7b). 21. PCR negative control. 22. DNA molecular weight markers (Appendix J) Comparing the sensitivity of C/O CEF s and DF-1 cells under different culture conditions The previous virus isolation sensitivity experiments reported on here were performed using 1% M199 medium for culture of both C/O CEF s and DF-1 cells. As DF-1 cells have been reported to grow best in Dulbecco's Modified Eagle Medium (DMEM), a comparison of the sensitivity of C/O CEF s and SPAFAS CEF s (grown in 1% M199) with DF-1 cells (grown in 1% DMEM) was performed using two different Australian isolates of ALV-J. Virus detection was performed using ALV-Ag ELISA. The results are presented in Table 4.5 SPAFAS CEF s again revealed a high level of background p27 expression making them unsuitable for virus propagation and interpretation of 14

26 results from these cells difficult. C/O CEF s were again found to be more sensitive than DF-1 cells for virus propagation, but the increased sensitivity under these culture conditions was reduced by 10 fold (compared to 1000 and 50 fold in previous experiments). The degree to which C/O CEF s have increased sensitivity for virus isolation has varied somewhat between different experiments. However, increased sensitivity for detection of ALV-J has been apparent in each of the separate experiments conducted. Given the consistency of this sensitivity data and the low level of endogenous p27 background as well as the absence of PCR background, C/O CEF s have been selected as the cells for virus isolation in these studies. Careful note was taken however that the presence of ALV-E in any field samples might also be propagated in C/O CEF s and contribute to a degree of positive reactions in ALV-Ag ELISA tests. The impact of isolating ALV-E will be considered in Section Table 4.5 Comparison of different cells using Australian isolates: Dilution SPAFAS CEF s C/O CEF s DF-1 cells UOM-210 UOM-224 UOM-210 UOM-224 UOM-210 UOM-224 neat control control control mean Note: results expressed in SP ratios Australian ALV-J isolates Table 4.6 on the following pages is a summary of all the field samples that were tested during this project. This summary includes virus isolations from various tissues, ALV-Ag testing, ALV-J antibody testing, PCR testing of tumour and feather DNA extracts. Tables 4.7, 4.8 and 4.9 are selected summaries of all the submissions. Table 4.7 includes the number of ALV-Ag positive samples found from each type of sample tested (tumours, whole blood, albumens and meconium). The whole blood samples have been further divided into those in which buffy-coat was used for virus isolation and those in which serum was used for virus isolation. The percentage of ALV-Ag positives compared to number of samples tested as well as the percentage of ALV-Ag positives that were identified by PCR (as either ALV-J or ALV-A) have also been calculated and included. This shows the highest percentage of Ag-ELISA positives identified by PCR to be 3.33 % from buffy-coat, 1.69 % from albumens and 0.10 % for serum. 15

27 Table 4.6 Summary of all submissions. Date Sample type No. of Samples Cultured Ab-ELISA Ag-ELISA PCR Ovarian Reference + ALV-J J98290/191 --/04/98 Tumour strain --/05/01 Myelocytoma 1 + ALV-J --/05/01 Myelocytoma 1 + ALV-J --/05/01 Myelocytoma 1 + ALV-J --/05/01 Myelocytoma 1 + ALV-J --/12/01 Whole blood 20 C/O bc 13/ / /15 + ALV-J --/12/01 Tumour 1 NA 1/1 + ALV-J --/12/01 Whole blood 20 C/O bc 3/20 + 3/20 + 3/3 + ALV-J --/12/01 Whole blood 20 C/O bc 3/20 + 3/20 + 3/3 + ALV-J --/12/01 Whole blood 20 C/O bc 16/19 + 2/20 + 4/5 + ALV-J 07/03/02 Albumens 21 C/O NA 21/ /21 + ALV-J 28/03/02 Albumens 10 C/O NA 10/10 + All negative 04/04/02 Whole blood 120 C/O bc 2/ /60 ALV-A 02/05/02 Serum 60 50/ /05/02 Whole blood 40 C/O bc All negative 08/05/02 Serum / / /05/02 Serum 120 All negative 26/ / /05/02 Albumens 67 C/O NA 27/67 + All negative 29/05/02 Serum / /06/02 Albumens 63 C/O NA 34/ /22 neg for ALV-J 26/06/02 Serum 49 All negative 11/ /07/02 Whole blood 43 C/O bc 9/43 + All negative 30/07/02 Meconium 99 C/O NA 1/99 + 1/1 + ALV-J 31/07/02 Serum 30 All negative 22/08/02 Serum 30 4/ /09/02 Whole blood C-swabs Feathers C/O bc 16 93/100 + NA NA 3/ /3 + ALV-J 16/09/02 Serum 30 13/ /09/02 Whole blood 50 C/O bc 3/50 + 3/3 + ALV-J 20/09/02 Tumours 2 NA NA 2/2 + ALV-J/A 02/10/02 Albumens 40 C/O NA All negative 02/10/02 Whole blood 48 C/O bc C/O bc C/O bc All negative All negative 7/24 + 2/12 + 7/12 + 7/ /16 + ALV-J 22/10/02 Albumens 320 C/O 53/320 + All negative 22/10/02 Albumens 67 C/O NA All negative 30/10/02 Whole blood 27 C/O bc 2/27 + 6/27 + 3/6 + ALV-J 07/11/02 Whole blood 70 C/O bc All negative 5/70 + 1/5 + ALV-J 14/11/02 Serum 100 4/ /11/02 Whole blood Feathers All negative NA

28 Date Sample type No. of Samples Cultured Ab-ELISA Ag-ELISA PCR 04/12/02 Whole blood 50 C/O bc 48/50 + 1/50 + 1/1 negative 27/12/02 Whole blood C-swabs Feathers C/O bc 2/29 + NA NA 8/29 + NA 09/01/03 Whole blood C-swabs Feathers 09/01/03 Whole blood C-swabs Feathers 09/01/03 Whole blood C-swabs Feathers C/O bc C/O bc C/O bc All negative NA NA 1/13 + NA NA 16/25 + NA NA All negative 2/13 + All negative 2/2 + ALV-J 2/2 + ALV-J 21/01/03 Tumours 2 C/O NA 2/2 + 29/01/03 Tumour 1 C/O NA + after culture ALV-J + tumour and culture 30/01/03 Tumour 1 C/O NA + after culture ALV-J + tumour and culture 04/02/03 Whole blood 60 C/O bc 21/ /21 + ALV-J 11/02/03 Whole blood 60 C/O bc 13/ / /03/03 Albumen 1 C/O NA + Albumen + after culture 19/03/03 Albumen 1 C/O NA + Albumen + after P1 Neg after P2 ALV-J + ALV-A negative Negative 19/03/03 Whole blood 30 C/O bc 3/30 + 1/30 + Negative 21/03/03 Whole blood 100 C/O bc All negative 1/100 + ALV-J + 27/03/03 Albumens 160 C/O NA All negative 27/03/03 Whole blood 24 C/O bc 3/24 + 5/24 + 1/5 + ALV-A 28/03/03 Whole blood 100 C/O bc 3/100 + All negative 02/04/03 Whole blood 62 C/O bc C/O bc 1/30 + 5/30 + 2/30 + All negative Negative Negative 03/04/03 Albumens 80 C/O NA 2/80 + 2/2 negative 08/04/03 Whole blood 90 C/O bc 10/ /90 + Negative 08/04/03 Whole blood 32 C/O bc 18/32 + 7/32 + 1/7 + ALV-A 08/04/03 Albumens 300 C/O NA 2/300 + Negative 09/04/03 Whole blood 31 C/O bc 24/ /31 + Negative 09/04/03 Whole blood 60 C/O bc C/O bc 17/ /30 + 6/30 + 4/30 + Negative 3/4 + ALV-A 09/04/03 Whole blood 30 C/O bc 17/30 + 6/30 + Negative 09/04/03 Whole blood 30 C/O bc 25/30 + 4/30 + 2/4 + ALV-A 16/04/03 Whole blood 60 C/O bc 29/ /60 + Negative 17/04/03 Whole blood 43 C/O bc 12/22 + 3/22 + 1/3 + ALV-A C/O bc 19/21 + 4/21 + Negative 30/04/03 Whole blood 60 C/O bc 32/60 + 9/60 + 1/9 + ALV-A 17

29 Date Sample type No. of Samples Cultured Ab-ELISA Ag-ELISA PCR 30/04/03 Whole blood 65 C/O bc C/O bc 32/ /30 + 2/32 + 2/33 + Negative Negative 30/04/03 Whole blood 90 C/O bc All Negative All negative 01/05/03 Whole blood 66 C/O bc C/O bc 11/ /32 + 3/34 + 4/32 + Negative 1/4 ALV-A 05/05/03 Whole blood 60 C/O bc C/O bc 21/ /30 + 1/30 + 2/30+ Negative Negative 15/05/03 Tumours 2 NA 2/2 + after 1/2 ALV-A culture 27/05/03 Whole blood 270 C/O s All negative 28/05/03 Whole blood 191 C/O s C/O s 2/63 + All negative Negative 04/06/03 Whole blood 180 C/O s 2/180 + Negative 12/06/03 Whole blood 270 C/O s 3/ /3 ALV-A 12/06/03 Whole blood 265 C/O s All negative 13/06/03 20/06/03 Whole blood 180 C/O s C/O s C/O s All negative All negative All negative 4/60 + 8/60 + 4/60 + Negative 1/8 ALV-A Negative 25/06/03 Whole blood 250 C/O s C/O s All negative All negative 27/06/03 Whole blood 300 C/O s All negative Note: C/O bc C/O CEF s inoculated with buffy-coat preparation in 24 well format. C/O s C/O CEF s inoculated with serum in 96 well format. = not tested. NA = not applicable ALV-J infection status in various Australian flocks. In a proportion of the total flocks tested during this study the infection status can be determined based on presence (+) and absence (-) of viremia (V) and antibody (A). Data is presented in Table 4.8. Of 278 birds tested, the overall frequencies of categories V+A-, V-A+, V+A- and V-A- were 30 (10.8%), 113 (40.6%), 22 (7.9%) and 113 (40.6%) respectively. The majority of birds tested from flock 3 (62.2%) and flock 5 (90.9%) belong to the no viremia with ALV-J antibody (V-A+) category. This data on infection profiles shows that the highest number of birds is found in the non-viremic antibody negative category (V-A- 40.6%) as compared to those viremic non-immune birds (V+A 10.8%). Like other exogenous ALV s, ALV-J is transmitted congenitally and horizontally. Chicks infected congenitally or horizontally soon after hatch will become permanently viremic, do not develop antibody and will shed virus into the environment throughout their lives. Other chickens infected horizontally after hatch develop antibody and become non viremic or a low level of infection may persist. Unlike the pattern found for viruses of other subgroups, horizontal transmission of ALV- J occurs very rapidly after hatch (Fadly and Smith, 1999) The high percentage of non-viremic immune birds recorded in this study indicates that the majority of birds were most likely being infected horizontally post-hatch. This situation is indicative of the presence of congenitally infected shedders, likely in very low numbers, that were transmitting horizontally within flocks. A summary analysis of the virological and serological survey shown in Table 4.9 (selected data only) reveals the presence of viremia in 31.2 % of flocks tested and antibody in 47.6 % of flocks. This indicates that infection is widespread in the flocks of some of the organisations tested in Australia. The prevalence of viremia ranged from 0-75 % and antibody ranged from 0-100%. Figure 4.6 depicts the SP ratio profiles from three different situations where antibody prevalences are 8, 50 and 96%. These charts demonstrate that different levels of SP ratio encountered when using the ALV-J Ab- 18

30 ELISA. Flocks with low percentage of birds with antibody had SP ratio s of less than 1.0. As the percentage of birds in a flock with antibody increases the SP ratio increases for 50 % of birds positive the SP ratio s are up to 1.6, for 96% up to 5.0. In a situation where a flock is >20 weeks old and has a reasonable level of antibody positive birds (>40%) and the SP ratio s are high (>2.0) then the Ab- ELISA will provide a useful tool for rapid screening of flocks in order to identify those in which virus isolation should be attempted. Table 4.7 Consolidated summary of ALV detection and isolation. Sample type Number Submitted Number Tested Number Ag ELISA positives ALV-J Detection PCR ALV-A detection PCR % of ALV-Ag positives Tumours na 10* Whole blood (6.37) Buffy-coat (11.80) Serum (1.15) nil Albumens (13.35) 19 nil 1.69 Meconiums (1.01) 1 nil 100 Note: Whole blood samples refers to those in which virus isolation was performed. A number of serum samples were submitted for ALV-J Ab-ELISA testing which are not included here. In the ELISA positive column figures in brackets represents the percentage positive compared to the number tested. ALV-A detection figures in this table represent ALV-A isolations. There are 16 co-isolations of ALV-A and ALV-J which have both been included in this column. *Tumour samples have been included in which PCR was positive from genomic extraction, even though virus isolation was not performed for every sample. 19

31 A positive negative B Samples C Figure 4.6 Antibody SP ratio variations. All charts are depicted with SP ratio plotted on the X axis and sample number on the Y axis. A. SP ratio plotted against sample number for a flock showing 8% Ab positive samples. B. SP ratio plotted against sample number for a flock showing 50% Ab positive samples. C. SP ratio plotted against sample number for a flock showing 96% Ab positive samples. Note: For ALV-J Ab-ELISA >0.6 is considered positive (0.6 cut off point is indicated on each plot) 20

32 Table 4.8 ALV-J infection status in various Australian flocks. Flock Genotype Age No tested Category V+A- V-A+ V+A+ V-A- 1 A (P) 1-day 10 2 (20.0) 1 (10.0) 1 (10.0) 6 (60.0) 2 A (P) 30 wk 20 2 (10.0) 2 (10.0) 1 (5.0) 15 (75.0) 3 B (P) 54wk 19 1 (5.3) 12 (63.2) 4 (21.1) 2 (10.5) 4 B (GP) 54 wk 20 5 (25.0) 3 (15.0) 10 (50.0) 2 (10.0) 5 A (P) 64 wk 99 1 (1.0) 90 (90.9) 2 (2.0) 6 (6.1) 6 A (P) 16 wk 27 5 (18.5) 1 (3.7) 1 (3.7) 20 (74.1) 7 A (P) 1 day 35 1 (2.9) 0 (-) 0 (-) 34 (97.1) 8 A (Br) 4 wk 12 2 (16.7) 0 (-) 0 (-) 10 (83.3) 9 A (P) 12 wk 12 7 (58.3) 0 (-) 0 (-) 5 (41.7) 10 A (P) 24 wk 12 1 (8.3) 2 (16.7) 3 (25.0) 6 (50.0) 11 A (P) 24 wk 12 3 (25.0) 2 (16.7) 0 (-) 7 (58.3) Total (10.8) 113 (40.6) 22 (7.9) 113 (40.6) GP: grand parent; P: parent; B: broiler; V: viremia; A: antibody Figures in the parenthesis indicate percentage Table 4.9 Prevalence of ALV-J and antibody in broiler breeder flocks. Genotype No flocks tested ALV-J isolation No of flocks positive No flocks tested ALV-J antibody No of flocks positive A (32.2) (52.9) B 2 2 (100.0) 2 2 (100) C 5 0 (-) 5 0 (-) D 1 0 (-) 1 0 (-) Total (31.25) (47.6) 21

33 4.13 C/O CEF s and DF-1: ALV-E background The propagation of ALV-E in C/O CEF s leads to an increased level of background when potential virus isolations are screened by ALV-Ag ELISA. Ninety-seven field whole blood samples that tested positive by Ag-ELISA after virus isolation from buffy-coat preparations in C/O CEF s were repassaged in DF-1 cells. These 97 samples were also screened by ALV-J and ALV-A specific PCR, of which 10/97 were identified as ALV-A alone (i.e. no ALV-J isolations). Figure 4.7 shows the SP ratio plotted against sample number for both C/O CEF s and DF-1 cells. Results for the DF-1 repassage reveal a decreased background level with only seven samples from the one hundred giving a positive SP ratio, these seven samples had been demonstrated to be ALV-A isolations by PCR after the initial isolation in C/O CEF s. Interestingly three samples also shown by PCR to be ALV-A isolations gave negative SP ratios after the repassage in DF-1 cells. This likely reflects a decreased sensitivity of DF-1 cells for infection by ALV-A, similar to that which had already been observed for ALV-J (Chapter 4). gs ELISA (C/O) SP ratio Sample 0.2 PCR negative ALV-A PCR positive gs ELISA (DF-1) Sample 0.2 PCR negative ALV-A PCR positive Figure 4.7 C/O vs DF-1 background with ALV-Ag ELISA. 22

34 4.14 Co-isolations of ALV-J and ALV-A In November 2002, to confirm the technology developed and the results being obtained in this project, 30 viral isolation samples from Australian field flocks were forwarded from the IAHL Melbourne to an ALV-J international reference centre, i.e. the Viral Oncogenesis group, Institute of Animal Health, Compton, UK. In March 2003 confirmation of results were received and are summarised in Table In 28/30 samples IAHL results concur with Compton. Two samples found by IAHL to be positive for ALV-J were unable to be cultured by Compton and this has been attributed to the prolonged storage of these samples. Of the 30 isolates checked in the UK, 16/30 contained a mixture of ALV-J plus subgroup-a. Five further p27-elisa positive samples identified by IAHL and submitted as ALV-J negative were confirmed to be negative also by Compton. These p27 positive reactions were most likely caused by ALV-E propagated in C/O CEF s but not by the C/E cells employed by Compton. Interestingly, one of the two single ALV-A isolates was negative for p27-elisa (Compton) but was still detected by PCR. Broiler breeders appear far less susceptible to infection with ALV-A than with ALV-J. The lower susceptibility is postulated to be the result of better clearance of ALV-A than of ALV-J by the immune system. Also, the lower genetic susceptibility to ALV-A of broiler breeders compared to layers results in lower horizontal and vertical transmission, this would explain why the prevalence of ALV-A in broiler breeders remains confined to incidental cases and has not resulted in serious field problems as has been the case with ALV-J. Very little has been published on the circulation of these viruses in broiler breeders, but the presence of ALV-A must be an additional cause for concern as an increase in transmission rates, possibly arising through continual circulation and selection pressure in the Australian industry, may occur along with the potential for forming A:J recombinants in the future. Considerably less concern is expressed at present, about the presence of ALV-A than for ALV-J, according to Australian industry specialists. However one source of ALV-A is likely to be imported genetic broiler stocks in that ALV-A has been detected by our laboratory in one recent importation prior to its release from quarantine. Advice from Dr. Venugopal K. Nair, Head Viral Oncogenesis group, Institute of Animal Health, Compton, UK indicates Co-occurrence of ALV-A and J is not uncommon in Europe. It depends on the line of chickens. Some of the chicken lines were clean from A. But there were many which had ALV-A as eradication was not strictly followed. So when the J emerged, they were co-infected. Although there is not much published on co-infection, I know that several European isolates are a mixture of A and J. Certainly the targets are different and we mostly hear about ML caused by ALV-J because of the higher frequency Recommendation Primary breeders should actively eliminate all replicating ALVs from their genetic breeding stocks as quickly as possible. Furthermore, all batches of genetic stocks imported in the future, should be screened to ensure their freedom from replicating ALVs. 23

35 Table 4.10 Confirmatory results for Australian samples (3.03) forwarded to the AFRC Compton, UK in November Sample no. p27-elisa IAHL p27-elisa Compton PCR UOM PCR Compton J Negative A+J A+J J Negative A+J A+J A+J A+J A+J A+J A+J A+J A+J A+J A+J A+J A+J A+J A+J A+J A+J A+J A A Negative Negative Negative Negative A A Negative Negative Negative Negative Negative Negative A+J A+J A+J A+J A+J A+J A+J A+J J J J J A+J A+J A+J A+J J J J J J J 24

36 5. Antigenic characterisation 5.1 Introduction Initial antigenic characterisation of isolates of ALV-J obtained in the USA showed that in the period between the isolation of the original ALV-J prototype HPRS-103 (Payne et al., 1991) and subsequent isolations (ADOL-Hc1 in 1993) substantial antigenic drift had occurred in that antibodies raised against HPRS-103 no longer recognised the newly isolated ADOL-Hc1. This drift most likely reflected immune responses exerting selective pressure that drives the antigenic variation. The antigenic characterisation of a number of Australian isolates to determine the degree of antigenic drift is described in this chapter. 5.2 The antigenic micro-neutralisation assay The antigenic relatedness of Australian ALV-J isolates was determined by a micro-neutralization assay using standard methods (Fadly and Witter, 1998). Briefly sera were diluted 1:5, mixed 1:2 with 100 units of virus, and incubated for 45 min at 37 C. Residual virus was assayed on C/O CEF s, cultured for seven days and tested for p27 antigen by ELISA. A negative test was considered evidence for antibody and indicated nearly complete neutralisation of the virus. Antisera against two reference strains (ADOL-Hc1 and J98290/191), and one field isolate (UOM-101), were tested against three reference strains (HPRS-103, ADOL-Hc1 and J98290/191) and four field isolates (UOM-101, UOM-202, UOM-217 and UOM-219) of ALV-J. 5.3 Antigenic variation is observed in Australian ALV-J isolates Table 5.1 shows the antigenic relationships among Australian and overseas isolates of ALV-J determined by the in vitro micro-neutralisation assay. Antigenic variation was observed among different isolates of ALV-J. Antibody to ADOL-Hc1, the US reference strain, neutralised HPRS-103, ADOL-Hc1, UOM-217 and UOM-219 but did not neutralise J98290/191, UOM-101, and UOM-202. Antibody to Australian reference strain J98290/191 neutralised two overseas reference strains (HPRS-103 and ADOL-Hc1) but did not neutralise UOM-101, UOM-217 and UOM-219. Antibody to Australian field isolate UOM-101 neutralised all strains except HPRS-103, UOM-217. The virus neutralisation assay results suggest that there is antigenic variation between overseas and Australian isolates and also among Australian isolates. Three Australian isolates (J98290/191, UOM-101 and UOM-202) were not neutralised by antibody to ADOL-Hc1, suggesting that these isolates and ADOL-Hc1 are not identical. The antibody to Australian reference strain (J98290/191) neutralised both UK and American reference viruses whereas antibody to Australian field isolate (UOM-101) failed to neutralise UK reference strain (HPRS-103). Venugopal et al. (1998) reported that ten of twelve ALV-J isolates tested were not neutralised by antibodies to any of ALV subgroups including J, and only two isolates were neutralised with a specific serum of HPRS-103, the prototype of ALV- J. Antibodies to ADOL-Hc1 neutralised HPRS-103, whereas antibody to HPRS-103 did not neutralise ADOL-Hc1 (Fadly and Smith, 1999). Antigenic variation among strains of ALV-J has been shown to be associated with changes in the envelope gene (Venugopal, et al., 1998; Silva, et al., 2000). Furthermore, the recent data suggest that this virus is in the process of continuous mutation (Silva and Fadly, 2000). This continual mutation and evolution of ALV-J isolates may seriously confound the development of effective diagnostic tests and vaccines. 25

37 Table 5.1 Virus micro-neutralisation assay results for various ALV-J isolates. Antiserum HPRS-103 ADOL-Hc1 J98290/ 191 Virus Strain UOM-101 UOM-202 UOM-217 UOM-219 ADOL-Hc J98290/ UOM

38 6. Experimental infection studies 6.1 Introduction A degree of antigenic drift has been demonstrated to occur in Australian isolates of ALV-J (Chapter 5). An experimental infection was designed to ascertain whether these Australian isolates have a similar pathogenicity to overseas isolates in light of the observed antigenic drift. 6.2 The viruses and chickens used ALV-J strains UOM-201 and UOM-224 isolated from Australian broiler parents were used in this study. These viral strains were originally isolated from buffy-coat samples and were identified as ALV-J by PCR. Broiler parent chicks (1 day old) hatched from a known ALV-J free broiler grand parent flock were obtained from a commercial breeding company. 6.3 The experimental design This experiment was conducted in the Animal Experimental Facility of University of Melbourne at Werribee. A total of 110 chicks were obtained from a commercial broiler breeding company immediately after hatch. All the chicks were individually identified by wing tags, weighed and swabbed for meconium. Five chicks were randomly separated and bled to confirm the ALV-J free status of the flock by virus isolation in C/O CEF culture and by ALV-J Antibody-ELISA. The remaining 105 chicks were randomly divided into three groups with 35 chicks each. Each group of chicks were housed in separate isolator equipped with HEPA air filters operating under negative pressure. Within four hours after hatch, chicks of group 1 and 2 were injected intra-peritoneally with ALV-J strains UOM-201 and UOM-224, respectively, at the dose rate of 10 5 TCID 50 per chick. Chicks in the group 3 were mock inoculated with M199 tissue culture medium to serve as controls. Experimental birds were reared to 26 weeks. Feed restriction and lighting programmes were followed as per the recommendations of the breeding company. 6.4 Sampling procedures Whole blood (with and without anticoagulant), cloacal swabs and feather pulp were collected from individual birds at 3, 6, 12, 18, and 26 weeks of age. Individual body weights were recorded at 3 and 6 weeks of age and vaginal swabs were obtained at 26 weeks of age. Five birds at 3, 6, 12 and 18 weeks of age and all the survivors at the end (26 weeks) were euthanised and weighed individually before undertaking necropsy examination. All the birds which died and were sacrificed during the experiment were necropsied for gross lesions, weights of bursa and spleens were recorded and various tissue samples were obtained for microscopic examination and detection of proviral DNA in the genomic DNA. Eggs were obtained group wise and albumen was separated for detecting shedding of virus and viral antigens. 6.5 Pathogenicity of Australian isolates Significant growth depression was noticed in ALV-J infected birds at 3 and 6 weeks of age as compared to control birds (Table 6.1). The effect of ALV-J on bursa and spleen weights is presented in tables 6.2 and 6.3 respectively. The mortality pattern among experimental groups is presented in Table 6.4. The most common neoplastic condition observed was myelocytoma. The other tumours noticed were renal adenoma, nephroblastoma and histiocytic sarcoma. The earliest age at which tumours were noticed was 6 weeks in the UOM-201 group and 12 weeks in the UOM-224 group. Myeloid tumours were characterised by skeletal myelocytomas affecting the inner sternum, neoplastic enlargement of liver, spleen, kidney, heart and trachea. Microscopically, the myeloid tumours consisted of immature granulated myelocytes, and were present as focal or diffuse 27

39 infiltrations in the affected organs. The status of viremia, antibody response and shedding of virus in infected birds at different ages is presented in Table 6.5. Table 6.1 Juvenile body weight of broilers infected with Australian strains of ALV-J Group Body weight (g) (Mean ± SD) Day old 3 weeks 6 weeks UOM ± 3.76 a ± a ± a UOM ± 4.61 a ± a ± a Control 46.2 ± 5.00 a ± b ± b a,b Means within a column having common superscripts do not vary significantly (p 0.05) Table 6.2 Ratios of bursa weights to body weights Group Bursa weight to body weight ratio x10, 000 (Mean ± SD) 6 weeks 12 weeks 18 weeks UOM ± 4.34 a 9.65 ± 3.15 a 5.26 ± 2.74 a UOM ± 3.24 b ± 3.62 b 8.94 ± 3.38 b Control ± 3.91 b ± 5.36 b 6.64 ± 0.75 a Organ (g)/body weight (g) X 10,000; values show average of five chickens. Values with in a column followed different lowercase superscript letters differ significantly (P<0.05). Table 6.3 Ratios of spleen weights to body weights Spleen weight to body weight ratio x 10,000 (Mean ± SD Group 6 weeks 12 weeks 18 weeks 26 weeks UOM ± 5.00 c ± 7.94 c ± 0.32 b ± a UOM ± 7.82 b ± 5.00 b ± 2.72 c ± 3.37 a Control ± 2.21 a 8.75 ± 1.35 a 9.08 ± 0.68 a 5.49 ± 1.65 b Organ (g)/body weight (g) X 10,000; values show average of five chickens. Values with in a column followed different lowercase superscript letters are significantly different (P<0.05). Table 6.4 Mortality pattern in experimentally infected birds at different ages Age Mortality (no) Group A Group B Group C 0-1 weeks 2/35 2/35 0/ weeks 1/33 1/33 0/ weeks 0/27 0/27 0/ weeks 1/22 1/22 1/ weeks 6/17 0/17 0/ weeks 0/5 0/7 0/8 28

40 Table 6.5 Infection Status of ALV-J infected birds at different ages Age UOM-201 UOM-224 Viremia Antibody Shedding Viremia Antibody Shedding 3 weeks 31/33 7/33 6/33 32/33 9/33 6/33 6 weeks 21/27 18/27 14/27 19/27 20/27 11/27 12 weeks 19/21 8/21 12/21 17/21 6/21 15/21 18 weeks 4/11 4/11 5/11 11/17 7/17 5/17 26 weeks 2/5 3/5 3/5 5/7 3/7 6/7 6.6 The advantages and disadvantages of PCR detection in feathers In order to address the questions about the suitability of feathers as a source of DNA for PCR detection, experimentally infected birds were utilised. The attraction of using feather pulp for the detection of ALV-J is the potential ease and speed of collection and detection and there is no need for any other equipment than a sterile 1.5 ml eppendorf tube per sample. Feathers also appear to be able to be stored for some time prior to processing (Davidson and Borenshtain, 2002). Results from feather sampling and PCR in our experimental infection were somewhat variable with only very small numbers of birds known to be positive for ALV-J being detected by feather PCR using the H5/ H7b primers described in Chapter 7 (feather samples were taken at 6, 12, 28 and 26 weeks). At the outset of this experiment, birds were infected intraperitoneally at 1 day old with 10 5 TCID 50 of virus per chick. This route of infection may have influenced the results obtained. Zavala et al. (2001) also carried out an experimental infection and compared PCR from feather pulp in embryo infected birds, birds infected intraperitoneally at three days old and in contact infected birds. They also found variable results particularly in three day old chicks and more so in contact infections. These authors also used a different set of primers for detection of proviral ALV-J in genomic feather extracts. These primers were designed to amplify the entire env region and were found to be more effective than the H5/H7 primer pair. During natural infection in the field situation (the closest experimental equivalent being contact infection in the isolator) there may be a low provirus concentration in the feather pulp, thus making it difficult to detect by PCR (Zavala et al., 2001). Thus, a potential draw back of the use of PCR for detecting ALV-J in feather pulp is that this assay may not be as sensitive as virus isolation under normal field conditions. The fact that we used intraperitoneal inoculation at 1 day old combined with using H5/H7b primers may explain the poor results achieved for feather PCR in our experimental infection. Another disadvantage of this approach is the fact that the absence of virus isolation limits PCR detection from feathers to only ALV-J and does not detect other ALV s that could potentially be present in Australian poultry. Sufficient feather pulp is also required in the feather for adequate DNA to be extracted, and this appears to occur optimally in a rather narrow age window between 1 and 7 weeks (Zavala et al., 2001). This is due to many feathers drying out (having low levels of moist pulp) as they move into less active growth periods. Thus, PCR directly from feather pulp cannot be recommended as a tool for reduction/eradication purposes but may be a useful tool for diagnosis in the research setting or if virus isolation procedures are not available. 29

41 7. Molecular biological detection of ALV-J 7.1 Introduction The polymerase Chain Reaction (PCR) is a procedure by which very small amounts of DNA can be converted into microgram amounts within a few hours. Specific primers (designed from published sequences) can be used to amplify pathogen specific DNA fragments from samples. There is no doubt about the value and potential of using molecular techniques in diagnostic virology. This does not apply solely to the detection and analysis of those viruses that cannot be isolated in cell cultures. Molecular amplification methods, predominantly PCR, offer such greatly increased sensitivity and specificity that their use is now amply justified for the detection of viruses such as ALV-J. Several reviews on the application of PCR in the diagnosis of poultry diseases have been published (Cavanagh, 1993; Cavanagh et al., 1997; Tripathy, 1998) 7.2 PCR detection of ALV-J Current methods for the detection of ALV-J in chickens include detection of viral group-specific antigen (p27) by an ELISA test (Smith et al., 1979; Clark and Doughert, 1980). However, this test is not suitable for the detection of antigen of purely exogenous ALV s, as the test will also detect endogenous viral p27 in certain samples (Crittenden and Smith, 1984). A common test for exogenous ALV in serum or tissue involves propagation of the virus in chicken embryo fibroblasts (CEF s), which are then disrupted and an ALV-Ag ELISA test performed (Fadly, 1989; Payne et al., 1992). Successful infection of CEF s will lead to the incorporation of proviral sequence into the genome of these cells and a PCR specific for this proviral DNA sequence can be used to detect the presence of ALV s. A PCR for the detection of viral RNA and proviral DNA from tissues infected with ALV-A have been described (van Woensel et al., 1990). This test used primers selected from the second and third variable regions of the gp85-env gene and is specific for ALV-A. PCR has also been used for the detection of vaccine contamination by ALV using primers designed for subgroups A to E (Hauptli et al., 1997). The env gene of ALV-J differs considerably from that of other subgroups and is believed to have evolved by recombination with a subfamily of endogenous retrovirus (Bai et al., 1995). The existence of endogenous elements in several lines of chickens, with a high degree of homology to the env gene of ALV-J, has the potential to interfere with the specific amplification of the env gene sequences of ALV-J. This necessitates the selection of primers that selectively amplify a region specific to exogenous ALV-J. The use of PCR for the detection of ALV-J is further complicated by the occurrence of antigenic variants among virus isolates with significant sequence changes in the env gene (Venugopal et al., 1998). The primer sequences used in this study (Table 7.1) for the specific amplification of ALV-J are derived from those initially described by Smith et al. (1998). The primer pair H5 and H7 were demonstrated to specifically amplify a 545 bp fragment of ALV-J. The primer H5 was designed against the 3 region of the pol gene that is conserved across several ALV subgroups (Figure 7.1). Primer H7 was designed from a conserved region of the gp85 sequence of a number of variant ALV-J viruses. Here we are using a modified version of H7 called H7b (personal communication Stewart Brown 2002) which has is a 1 bp shift in an attempt to improve the specificity of this primer. 7.3 PCR positive and negative controls A number of essential controls are necessary in order to confidently determine the presence or absence of ALV-J in C/O CEF cultures inoculated from a field sample. These controls are needed to demonstrate: Adequate sensitivity of viral isolation/propagation in C/O CEF s (a serial dilution of viable virus, usually ALV-A, is required for this tissue culture positive control). 30

42 Absence of contamination in cultured cells (the culture and extract DNA from uninfected C/O CEF s in conjunction with virus isolation from field samples is used as a tissue culture negative control). Adequate sensitivity/confirmation of the PCR reaction (a serial dilution of a previously extracted ALV-J is used as a PCR positive control). Absence of contamination in PCR reagents (all PCR reagents are included in a reaction minus the template for the PCR negative control). In the case of a negative result for ALV-J, a positive control that demonstrates the presence of amplifiable genomic DNA (a set of primers towards a known chicken sequence with a similar sensitivity as the ALV-J primers are used for a DNA extraction positive control; a set of β-actin primers is used for this purpose). In some circumstances specificity can be demonstrated more confidently by including other ALV subgroups as a negative control (usually ALV-A/perhaps ALV-B; when used, a positive control for the presence of these subgroups also becomes necessary). Depending on the result obtained each of these controls becomes more or less critical. For example, if virus is demonstrated to be present in all samples (or can be easily isolated), then the tissue culture positive control becomes less critical while the tissue culture negative is essential to eliminate the possibility of a laboratory contamination. Similarly if one wishes to declare a flock free from ALV-J (because no virus was able to be isolated) it becomes critical to demonstrate both the level of sensitivity of the isolation process (tissue culture positive) and the PCR reaction (PCR positive) and the integrity of the DNA isolation process (DNA extraction positive). Suspected co-infections with more than one subgroup of ALV can be further investigated using more than one subgroup of ALV to increase the confidence of the PCR specificity. 31

43 Table 7.1 Primer sequences and specificity. Primer Sequence (5-3 ) Specificity/use Pair Product H5 5 -GGATGAGGTGACTAAGAAAG-3 Targets the 3 pol gene in all ALV a universal ALV primer. Various Various H7 5 -CGAACCAAAGGTAACACACG-3 Specific for subgroup J binds in env gp85 H5 545bp H7b 5 -GAACCAAAGGTAACACACGT-3 Improved version of H7 (shifted). Specific for subgroup J binds in env gp85 H5 544bp Env -A 5 -AGAGAAAGAGGGGYGTCTAAGGAGA-3 Specific for subgroup A binds in env gp85 H5 694bp AD1 5 -GGGAGGTGGCTGACTGTGT-3 ALV subgroups A-E binds in gp85 H5 360bp Bar 5 -CACAACCCACACGCAGCCCTG-3 Amplifies part of the chicken β-actin gene. A positive control for chicken genomic DNA Baf 401bp Baf 5 -TCTGGTGGTACCACAATGTACCCT-3 Amplifies part of the chicken β-actin gene. A positive control for chicken genomic DNA Bar 401bp J3 5 -TATTGCTGTTTCATCGTTA-3 Primer used to amplify the env region of ALV-J J5 ~2124bp J5 5 -GTGCGTGGTTATTATTTCC-3 Primer used to amplify the env region of ALV-J J3 ~2124bp 7.4 The primer pair H5 and H7b are specific for ALV-J The original characterisation of the primer pair H5/H7 and demonstration of their specificity for ALV- J was performed by Smith et al. (1998). However, given the slight modification of H7 to H7b and the use of Australian C/O CEF s for virus isolation and propagation it is necessary to demonstrate that the primer pair H5 and H7b is specifically amplifying a product from ALV-J and not from other subgroups of ALV or from endogenous sequences present in the Australian C/O CEF s. The sequence, specificity and predicted product of all primers used in this study are listed in Table 7.1 and depicted in Figure 7.1 Representative ALV subgroups A, B, C, D, E and J were propagated in C/O CEF s (Appendix K) and the genomic DNA extracted (Appendix H). Each subgroup was subjected to PCR with H5/H7b (designed to be specific for ALV-J) and also H5/AD1 (this primer pair amplifies a fragment from subgroups A, B, C, D, and E but not from ALV-J). Figure 7.2 demonstrates that the primer pair H5/H7b only amplify a product from C/O CEF s infected with ALV-J (HPRS-103). An amplification product with H5/AD1 for ALV-A, B, C, D and E confirms these virus were propagated and are not amplified with H5/H7b under the conditions used (Appendix F). The absence of a PCR product from uninfected C/O CEF s by either H5/H7b or H5/AD1 confirms the lack of any endogenous sequences in these cells that may interfere with the specificity of this ALV-J PCR. 32

44 LTR gag pro env LTR env H bp env region J5 AD1* H7b Env-A J3 544 bp ALV-J 694 bp ALV-A (H5/env- 360 bp ALV s (H5/AD1) *AD1 primer recognises ALV sub-groups A, B, C, D (but not J) Figure 7.1 Schematic of PCR primers and products. 7.5 The primer pair H5 and env-a are specific for ALV-A The primer pair H5/env-A was designed to specifically amplify the env region from ALV-A. These primers are important because ALV-A is likely to be circulating in the Australian poultry industry and it thus becomes important to be able to distinguish between ALV-J and ALV-A after propagation in C/O CEFs. This is readily achieved using PCR (but is not possible by ALV-Ag ELISA). Representative ALV subgroups A, B, C, D, E and J were propagated in C/O CEF s (Appendix K) and the genomic DNA extracted (Appendix H). Each subgroup was subjected to PCR with H5/env-A and with H5/AD1 (this primer pair amplifies a fragment from subgroups A, B, C, D, and E but not from ALV-J) or H5/H7b (to confirm the presence of ALV-J). Figure 7.3 demonstrates that the primer pair H5/env-A only amplify a product from C/O CEF s infected with ALV-A (RSV-A). An amplification product with H5/AD1 for ALV-B, C, D and E confirms these viruses were propagated and are not amplified with H5/env-A under the conditions used (Appendix F). The absence of a PCR product from uninfected C/O CEF s by either H5/env-A or H5/AD1 confirms the lack of any endogenous sequences in these cells that may interfere with specificity of this ALV-A PCR. 7.6 The isolation and propagation of ALV-J from field samples The propagation of ALV-J in C/O CEF s requires inoculation with viable virus, the under lying nature of an ALV-J infection enables the isolation and propagation of ALV-J from a number of different tissues. Albumen, blood, feathers, tumours tissue and meconium have all been demonstrated to contain viable virus for isolation. We set out to isolate and propagate ALV-J from each of these tissues. The procedure for handling each sample and subsequent culturing is described in Appendices K and A. Figure 7.4 demonstrates the successful isolation of ALV-J in each of these tissues from field samples. 7.7 ALV-J PCR with DNA extracted from tumours and feathers The propagation of ALV-J in C/O CEF s from field samples requires tissue culture facilities and two passages in culture (10 days in total, one passage of 7 days is also possible see Chapter 9) to confirm the presence of incorporated proviral DNA by specific PCR. Direct extraction of genomic DNA from 33

45 both tumour and feather tissue is appealing because it requires less time to detect ALV-J infection. Genomic DNA was extracted from a number of suspect tumours as well as from feathers (extraction procedures in Appendix H) and subjected to PCR with H5/H7b primers. Figure 7.3 is an example of successful amplification of ALV-J sequence from both these sources of genomic DNA. The type and nature of tumours can often be indicative of ALV-J infection, thus PCR of genomic material from these tumours may only be useful in supporting an already reasonably definitive diagnosis. Furthermore, given the usual late appearance of tumours, the use of tumours for early detection by PCR is likely to be limited. The use of feathers for PCR diagnosis may well hold greater promise. The ability to detect ALV-J infection from feathers has been discussed (Chapter 5). 7.8 Conclusions Results presented in this chapter demonstrate that the molecular biological techniques which are suitable for the detection of ALV-J (and ALV-A) have been established and can now be made available to the Australian poultry industry. Figures 7.2 and 7.3 demonstrate that primers for both ALV-J (H5/H7b) and ALV-A (H5/env-A) are specific for both these viruses; no spurious or nonspecific amplification was observed for any other ALV sub-groups (A, B, C, D and E when using H5/H7b or B, C, D, E and J when using H5/env-A). Just as importantly, no spurious or non-specific amplification was observed with uninfected C/O CEF s indicating the absence of endogenous sequences that may cross-hybridise with these PCR primers. This combined with data from the sensitivity assays (Chapter 4) confirms the suitability and superiority of C/O CEF s for virus isolation. The fact that C/O CEF s are able to propagate all sub-groups of ALV (including ALV-E) is also considered an advantage as these cells are more likely to propagate any new variant forms of ALV that may arise. The availability of DF-1 cells (which do not allow the propagation of ALV-E) are a useful secondary tool to distinguish between exogenous and endogenous viral isolates (Section 4.13). Our results clearly demonstrate that both these primer sets H5/H7b and H5/env-A are specific for ALV-J and ALV-A respectively and can be readily used to detect these viruses. Furthermore, H5/H7b primers have been used to demonstrate the successful isolation of ALV-J from a number of different Australian field tissue samples. These tissues include tumours, feathers, buffy-coat, albumen and meconium (Figure 7.4). These primers are therefore the principal tool for confirming the isolation of ALV-J (or ALV-A). 34

46 bp 401 bp Figure 7.2 Specificity of the primers H5/H7b. All exogenous ALV s were cultured in C/O CEF s and the genomic DNA extracted and subject to PCR with H5/H7b primers. Only ALV-J was amplified using these primers (lane 7). The presence of ALV-A, B, C, D and E were confirmed by the amplification of ~360 bp fragment with the primers H5/AD1 (lanes 10-14). All negatives control showed no amplification. 1. DNA Molecular weight markers. 2. ALV-A (H5/H7b). 3. ALV- B (H5/H7b). 4. ALV-C (H5/H7b). 5. ALV-D (H5/H7b). 6. ALV-E (H5/H7b). 7. ALV-J (H5/H7b). 8. Uninfected C/O CEF s (H5/H7b). 9. PCR negative control (H5/H7b). 10. ALV-A (H5/AD1). 11. ALV-B (H5/AD1). 12. ALV-C (H5/AD1). 13. ALV-D (H5/AD1). 14. ALV-E (H5/AD1). 15. ALV-J (H5/AD1). 16. Uninfected C/O CEF s (H5/AD1). 17. PCR negative control (H5/AD1). 18 DNA Molecular weight markers bp 544 bp 401 bp Figure 7.3 Specificity of the primers H5/Env-A. All exogenous ALV s were cultured in C/O CEF s and the genomic DNA extracted and subject to PCR with H5/Env-A primers. Only ALV-A (lane bp fragment) was amplified using these primers. The presence of ALV-B, C, D and E were confirmed by the amplification of ~360 bp fragment with the primers H5/AD1 (lanes 11-14). The presence of ALV-J was confirmed with H5/H7b primers (544 bp fragment lane 15). All negatives control showed no amplification. 1. DNA Molecular weight markers. 2. ALV-A (H5/Env-A). 3. ALV-B (H5/Env-A). 4. ALV-C (H5/Env-A). 5. ALV-D (H5/Env-A). 6. ALV-E (H5/Env-A). 7. ALV-J (H5/Env-A). 8. Uninfected C/O CEF s (H5/Env-A). 9. PCR negative control (H5/Env-A). 10. ALV-A (H5/AD1). 11. ALV-B (H5/AD1). 12. ALV-C (H5/AD1). 13. ALV-D (H5/AD1). 14. ALV-E (H5/AD1). 15. ALV-J (H5/H7b). 16. Uninfected C/O CEF s (H5/AD1). 17. PCR negative control (H5/AD1). 18. DNA Molecular weight markers. 35

47 bp Figure 7.4 ALV-J PCR on isolates from different tissues. A range of ALV-J isolates from a number of different tissues were amplified using ALV-J specific primers H5/H7b (lanes 3-7). Uninfected C/O CEF s demonstrated no amplification with H5/H7b (lane 8). The presence of genomic DNA in this sample was confirmed by the amplification of a 401 bp β-actin fragment with Baf/Bar primers (lane 9). No amplification of ALV-A or B sequences occurred with H5/H7b (lanes 10 and 12). The presence of these viruses was confirmed by the amplification of ~360 bp fragment with H5/AD1 (lanes 11 and 13). All other negative controls showed no amplification. 1. DNA Molecular weight markers. 2. ALV-J positive control (H5/H7b). 3. Tumour sample extract (H5/H7b). 4. Feather sample extract (H5/H7b). 5. Buffy coat isolation (H5/H7b). 6. Albumen isolation (H5/H7b). 7. Meconium isolation (H5/H7b). 8. Uninfected C/O CEF s (H5/H7b). 9. Uninfected C/O CEF s (Baf/Bar). 10. ALV-A control (H5/H7b). 11. ALV-A (H5/AD1). 12. ALV-B (H5/H7b). 13. ALV-B (H5/AD1). 14 PCR negative (H5/H7b). 15. PCR negative (H5/AD1). 16. PCR negative (Baf/Bar). 17. ALV-J positive control (no primers). 18. DNA Molecular weight markers. 36

48 8. Molecular biological analysis of ALV-J 8.1 Introduction Sequence analysis studies suggest that ALV-J has arisen through genetic recombination between endogenous and exogenous avian leukosis viruses (Bai et al., 1995). The virus has also been demonstrated to have a high level of antigenic drift (Venugopal et al., 1998), leading to sequence variability amongst isolates. This variability is useful when trying to determine the relationship between different isolates. Sequence analysis of Australian isolates of ALV-J can be used to determine the genetic variation and phylogenetic relationship of different isolates. The envelope region of ALV-J is the important site of nucleotide variation as this leads to variation in the proteins present on the surface of the virus and ultimately to antigenic variation. PCR can be used to specifically amplify the entire env region, which can then be cloned and sequenced. This approach will enable the determination of the complete envelope nucleotide sequence from primary sequence data. 8.2 PCR amplification of ALV-J env region The primers J3 and J5 have been designed to amplify an ~2124 bp fragment of ALV-J containing the entire sequence for gp85 and tm37 (see Table 7.1 and Figure 7.1). Figure 8.1 shows the successful amplification of a ~2124 bp fragment from a number of different Australian isolates. Each of these amplified products shows a slight variation in migration through the agarose gel indicating the heterogeneous nature of the different envelope sequences. This indicates at the gross level a degree of sequence variation occurring in these Australian isolates of ALV-J bp 544 bp Figure 8.1 ALV-J envelope PCR. A number of Australian ALV-J isolates were propagated in C/O CEF s and the genomic DNA extracted and subject to PCR with J5 and J3 primers. These primers amplify a ~2124 bp fragment of the env region of ALV-J. This amplified fragment contains the entire coding region for gp85 and tm DNA Molecular weight markers. 2. ALV-J control HPRS-103 (H5/H7b). 3. ALV-J control HPRS-103 (J5 /J3 ). 4.Australian isolate UOM 201 (J5 /J3 ). 5. Australian isolate UOM 216 (J5 /J3 ). 6. Australian isolate UOM 219 (J5 /J3 ). 7. Australian isolate UOM 224 (J5 /J3 ). 8. Australian isolate J98290/191 (J5 /J3 ). 9. Uninfected C/O CEF s. (J5 /J3 ) 10. PCR negative control (J5 /J3 ). 11. DNA Molecular weight markers. 37