avian leukosis virus sub-group j (alv-j) leukosis virus sub-group j (alv-j) developing laboratory...

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

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© 2004 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 024 4 ISSN 1440-6845 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 02 6272 3186. Researcher Contact Details T. J. Bagust The University of Melbourne Faculty of Veterinary Science Cnr Park Drive and Flemington Rd. Parkville, Victoria 3010. Phone: 03 8344 9675 Fax: 03 8344 9676 Email: [email protected] 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: 02 6272 4539 Fax: 02 6272 5877 Email: [email protected] Website: http://www.rirdc.gov.au Published in July 2004 Printed on environmentally friendly paper by Canprint

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Foreword The ALV-J subgroup of avian leukosis virus has emerged in recent years as causing tumours and mortalities in 25-55 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 1998-99 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 www.rirdc.gov.au/reports/Index.htm

purchases at www.rirdc.gov.au/eshop

Simon Hearn Managing Director Rural Industries Research and Development Corporation

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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.

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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

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Contents Foreword..............................................................................................................................................................III

Acknowledgments ............................................................................................................................................... IV

Abbreviations ....................................................................................................................................................... V

List of figures and tables .................................................................................................................................VIII

Executive Summary ............................................................................................................................................IX

1. Introduction....................................................................................................................................................... 1

2. Objectives........................................................................................................................................................... 1

3. Background ....................................................................................................................................................... 1 3.1 Introduction .................................................................................................................................................. 1 3.2 The Avian Leukosis Viruses (ALV's)........................................................................................................... 2 3.3 Recognition, origin and evolution of ALV-J................................................................................................ 3 3.4 Diseases, pathogenesis and effects on production ........................................................................................ 4 3.5 Transmission and shedding of ALV infections ............................................................................................ 5 3.6 Recent publications....................................................................................................................................... 6

4. Virology and Serology Results ......................................................................................................................... 7 4.1 Introduction .................................................................................................................................................. 7 4.2 Avian Leukosis Virus Antigen test kit.......................................................................................................... 7 4.3 Avian Leukosis Virus sub-group J Antibody test kit.................................................................................... 7 4.4 Viral prototypes ............................................................................................................................................ 8 4.5 Choosing cells for propagation of ALV-J. ................................................................................................... 8 4.6 Determining endogenous p27 levels in proposed cell lines.......................................................................... 8 4.7 PCR characterisation of uninfected cell lines ............................................................................................... 9 4.8 Comparison of cells for sensitivity of ALV-J propagation using a viral tissue culture

viral stock ................................................................................................................................................... 11 4.9 Sensitivity assay using a buffy-coat viral stock.......................................................................................... 13 4.10 Comparing the sensitivity of C/O CEF’s and DF-1 cells under different culture

conditions ................................................................................................................................................. 14 4.11 Australian ALV-J isolates......................................................................................................................... 15 4.12 ALV-J infection status in various Australian flocks................................................................................. 18 4.13 C/O CEF’s and DF-1: ALV-E background .............................................................................................. 22 4.14 Co-isolations of ALV-J and ALV-A ........................................................................................................ 23 4.15 Recommendations..................................................................................................................................... 23

5. Antigenic characterisation ............................................................................................................................. 25 5.1 Introduction ................................................................................................................................................ 25 5.2 The antigenic micro-neutralisation assay.................................................................................................... 25 5.3 Antigenic variation is observed in Australian ALV-J isolates.................................................................... 25

6. Experimental infection studies....................................................................................................................... 27 6.1 Introduction ................................................................................................................................................ 27 6.2 The viruses and chickens used.................................................................................................................... 27 6.3 The experimental design............................................................................................................................. 27 6.4 Sampling procedures .................................................................................................................................. 27 6.5 Pathogenicity of Australian isolates ........................................................................................................... 27 6.6 The advantages and disadvantages of PCR detection in feathers ............................................................... 29

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7. Molecular Biological detection of ALV-J...................................................................................................... 30 7.1 Introduction ................................................................................................................................................ 30 7.2 PCR detection of ALV-J............................................................................................................................. 30 7.3 PCR positive and negative controls ............................................................................................................ 30 7.4 The primer pair H5 and H7b are specific for ALV-J.................................................................................. 32 7.5 The primer pair H5 and env-A are specific for ALV-A. ............................................................................ 33 7.6 The isolation and propagation of ALV-J from field samples ..................................................................... 33 7.7 ALV-J PCR with DNA extracted from Tumours and Feathers .................................................................. 33 7.8 Conclusions ................................................................................................................................................ 33

8. Molecular Biological analysis of ALV-J........................................................................................................ 37 8.1 Introduction ................................................................................................................................................ 36 8.2 PCR amplification of ALV-J env region .................................................................................................... 37 8.3 Cloning and sequencing of the env region.................................................................................................. 38 8.4 Sequence analysis and phylogeny .............................................................................................................. 38 8.5 Sequence comparisons................................................................................................................................ 38 8.6 Phylogenetic analysis of Australian isolates............................................................................................... 39 8.7 Conclusions ................................................................................................................................................ 40

9. Development of practical methodologies for the detection of ALV-J......................................................... 41 9.1 Introduction ................................................................................................................................................ 41 9.2 A 96 well culture format of C/O CEF’s for virus isolation ........................................................................ 41 9.3 A comparison of 24 well format vs 96 well format. ................................................................................... 41 9.4 Comparison of 96 and 24 well formats in field samples ............................................................................ 42 9.5 What Samples are recommended?.............................................................................................................. 42

10. Implications and recommendations............................................................................................................. 44

11. References ...................................................................................................................................................... 45

12. Appendices..................................................................................................................................................... 49

Appendix A: Sample preparation and handling. ............................................................................................. 51

Appendix B: Precautions to avoid PCR contaminations................................................................................. 52

Appendix C: Collection of blood for ALV Monitoring.................................................................................... 53

Appendix D: C/O CEF Micro-culture for monitoring of ALV’s. ................................................................... 54

Appendix E: Interpretation and calculations for ALV-Ag and ALV-J Antibody test. ............................... 57

Appendix F: ALV-J PCR conditions and procedure....................................................................................... 59

Appendix G: Buffy-coat preparation. ............................................................................................................... 60

Appendix H: Genomic DNA extraction from C/O CF’s and Feathers. ......................................................... 61

Appendix I: Preparation of C/O CEF’s. ........................................................................................................... 62

Appendix J: DNA molecular weight markers. ................................................................................................. 65

Appendix K: The 24 well culture procedure. ................................................................................................... 66

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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. 4.10 Confirmation results for Australian samples (3.03) forwarded to the AFRC Compton, UK in November 2002. 5.1 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-Hc1. 9.1 Sensitivity for detection of ALV-J and ALV-A in C/O CEF culture in 96 well format (+/- DEAE) versus 24 well format.

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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 1998. 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 2000-2003 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

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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 1000 - 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 2002-03, 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 2000-02. 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 egg-transmission 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

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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).

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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 1999. 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 sub-optimal 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).

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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 sub-optimal broiler productivity in Australia during 1998-99 (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.

ALV-J (7841 bp)

gag pol env

CA p27 NC p12

gp 85 SU

gp 37 TM

RT p68 IN p32 PR p15

Avian Leukosis Virus sub-group J (ALV-J):

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.

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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 20-30 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 NDV), absence of a proof-reading mechanism for ALV-

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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 92-98.8%. 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 25-30 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 25-55 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

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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 1996-98 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.

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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+S-and 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., 2002. See Section D).

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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 5007.00) 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 5007.02) 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 12-14 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

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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 Proviral DNA extracted and used for a PCR control.

RSV-A

ALV-A Viral stock used routinely as a tissue culture positive for viral isolation

RAV-2

ALV-B Proviral DNA extract and used for a PCR control

RAV-49

ALV-C Proviral DNA extract and used for a PCR control

RAV-50

ALV-D Proviral DNA extract used for PCR control

RAV-

ALV-E Proviral DNA extract used for PCR control

ADOL-Hc1

ALV-J USA prototype ALV-J isolate. Proviral DNA extract used for PCR control

ADOL-7501

ALV-J A USA isolate which is a transforming/oncogenic virus. Proviral DNA extract used for PCR control

HPRS-103

ALV-J The original ALV-J prototype from the UK. Proviral DNA extract used for PCR control

J98290/191

ALV-J 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-12203 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

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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 0.01. 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-1 0.09 0.07

C/O CEF’s P-2 0.08 0.06

C/O CEF’s P-3 0.08 0.06

C/O CEF’s P-4 0.09 0.07

SP Mean 0.06

DF-1 cells P-1 0.05 0.01

DF-1 cells P-2 0.05 0.01

DF-1 cells P-3 0.04 0.00

DF-1 cells P-4 0.05 0.01

SP Mean 0.01

SPF CEF’s P-1 0.22 0.32

SPF CEF’s P-2 0.22 0.32

SPF CEF’s P-3 0.23 0.33

SPF CEF’s P-4 0.22 0.32

SP Mean 0.32

Kit Positive control 0.60 1.00

Kit Positive control 0.61 1.00

SP Mean 1.00

Kit Negative control 0.04 0.00

Kit Negative control 0.04 0.00

SP Mean 0.00

4.7 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

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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).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

544 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).

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1 2 3 4 5 6 7 8 9 10

694 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

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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

10-1 1.90 2.59 1.61 10-2 0.90 1.65 0.44 10-3 0.25 1.06 0.50 10-4 0.23 0.30 0.44 10-5 0.00 0.21 0.51 10-6 -0.03 0.21 0.49 10-7 -0.04 0.24 0.47 10-8 -0.04 0.19 0.54

Control -0.04 0.17 0.50 Note: positive SP ratios are bolded and control represents uninfected cells.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 SPF CEF’s C/O CEF’s DF-1

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). 3. 10-1 SPAFAS CEF’s (H5/H7b). 4. 10-2 SPAFAS CEF’s (H5/H7b). 5. 10-3

SPAFAS CEF’s (H5/H7b). 6. 10-4 SPAFAS CEF’s (H5/H7b). 7. 10-5 SPAFAS CEF’s (H5/H7b). 8. 10-6 SPAFAS CEF’s (H5/H7b). 9. 10-6 SPAFAS CEF’s (H5/H7b). 10. 10-1 C/O CEF’s (H5/H7b). 11. 10-2 C/O CEF’s (H5/H7b). 12. 10-3 C/O CEF’s (H5/H7b). 13. 10-4 C/O CEF’s (H5/H7b). 14. 10-5 C/O CEF’s (H5/H7b). 15. 10-6 C/O CEF’s (H5/H7b). 16. 10-1 DF-1 cells (H5/H7b). 17. 10-2 DF-1 cells (H5/H7b). 18. 10-3 DF-1 cells (H5/H7b). 19. 10-4 DF-1 cells (H5/H7b). 20. 10-5 DF-1 cells (H5/H7b).

13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

SPF CEF’sC/O CEF’s DF-121 22 23 24 25 26 27 28 29 30

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). 2. 10-1 C/O CEF’s (H5/H7b). 3. 10-2 C/O CEF’s (H5/H7b). 4. 10-3 C/O CEF’s (H5/H7b). 5. 10-4 C/O CEF’s (H5/H7b). 6. 10-5 C/O CEF’s (H5/H7b). 7. 10-6 C/O CEF’s (H5/H7b). 8. 10-7 C/O CEF’s (H5/H7b). 9. 10-8 C/O CEF’s (H5/H7b). 10. Uninfected C/O CEF’s (H5/H7b). 11. 10-1 SPAFAS CEF’s (H5/H7b). 12. 10-2 SPAFAS CEF’s (H5/H7b). 13. 10-3 SPAFAS CEF’s (H5/H7b). 14. 10-4 SPAFAS CEF’s (H5/H7b). 15. 10-5 SPAFAS CEF’s (H5/H7b). 16. 10-6 SPAFAS CEF’s (H5/H7b). 17. 10-7 SPAFAS CEF’s (H5/H7b). 18. 10-8

SPAFAS CEF’s (H5/H7b). 19 Uninfected SPAFAS CEF’s (H5/H7b). 20. 10-1 DF-1 cells (H5/H7b). 21. 10-2 DF-1 cells (H5/H7b). 22. 10-3 DF-1 cells (H5/H7b). 23. 10-4 DF-1 cells (H5/H7b). 24. 10-5 DF-1 cells (H5/H7b). 25. 10-6 DF-1 cells (H5/H7b). 26. 10-7 DF-1 cells (H5/H7b). 27. 10-8 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.

14

Table 4.4 Isolation of ALV-J from buffy-coat in C/O CEF’s and DF-1 cells.

SP ratio

Dilution C/O CEF’s DF-1 cells

10-1.0 1.68 0.87 10-1.5 1.53 0.12 10-2.0 1.00 0.07 10-2.5 0.31 0.02 10-3.0 0.20 -0.01 10-3.5 0.05 -0.01 10-4.0 0.03 -0.02

Control 0.03 -0.01 Note: positive SP ratio’s are bolded and control represents uninfected cells.

1 2 3 4 5 6 7 8 9 10111213 1415 1617181920

C/O CEF’sDF-1 2122

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 10-1.0 and 10-2.5 for C/O CEF's. 1. DNA molecular weight markers (Appendix J). 2. ALV-J positive PCR control. 3. 10-1.0 DF-1 cells (H5/H7b). 4. 10-1.5 DF-1 cells (H5/H7b). 5. 10-2.0 DF-1 cells (H5/H7b). 6. 10-2.5 DF-1 cells (H5/H7b). 7. 10-3.0 DF-1 cells (H5/H7b). 8. 10-3.5 DF-1 cells (H5/H7b). 9. 10-4.0 DF-1 cells (H5/H7b). 10. Uninfected DF-1 cells (H5/H7b). 11. PCR negative control (H5/H7b). 12. ALV-J positive PCR control. 13. 10-1.0 C/O CEF’s (H5/H7b). 14. 10-1.5 C/O CEF’s (H5/H7b). 15. 10-2.0 C/O CEF’s (H5/H7b). 16. 10-2.5 C/O CEF’s (H5/H7b). 17. 10-3.0 C/O CEF’s (H5/H7b). 18. 10-3.5 C/O CEF’s (H5/H7b). 19. 10-4.0 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). 4.10 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

15

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 4.13.

Table 4.5 Comparison of different cells using Australian isolates:

SPAFAS CEF’s C/O CEF’s DF-1 cells

Dilution UOM-210 UOM-224 UOM-210 UOM-224 UOM-210 UOM-224

neat 2.084 1.954 2.985 1.785 2.753 1.758 10-1 1.519 1.344 2.055 1.241 1.492 0.917 10-2 1.008 0.751 2.516 0.777 0.862 0.561 10-3 0.334 0.270 0.320 0.199 0.096 0.068 10-4 0.246 0.228 0.119 0.096 0.016 0.021 10-5 0.241 0.222 0.087 0.084 0.008 0.010 10-6 0.201 0.203 0.082 0.082 0.005 0.010 10-7 0.196 0.199 0.098 0.071 0.011 0.008 10-8 0.206 0.201 0.092 0.084 0.008 0.008 10-9 0.203 0.203 0.079 0.079 0.008 0.008 10-10 0.199 0.201 0.100 0.087 0.008 0.010 control 0.201 0.199 0.077 0.084 0.010 0.011 control 0.199 0.201 0.082 0.084 0.008 0.008 control mean 0.200 0.082 0.009

Note: results expressed in SP ratios.

4.11 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.

16

Table 4.6 Summary of all submissions.

Date

Sample type

No. of Samples

Cultured

Ab-ELISA

Ag-ELISA

PCR

--/04/98

Ovarian Tumour

J98290/191

Reference strain

ND ND + ALV-J

--/05/01 Myelocytoma 1 ND ND + ALV-J --/05/01 Myelocytoma 1 ND ND + ALV-J --/05/01 Myelocytoma 1 ND ND + ALV-J --/05/01 Myelocytoma 1 ND ND + ALV-J --/12/01 Whole blood 20 C/O bc 13/20 + 15/20 + 15/15 + ALV-J --/12/01 Tumour 1 ND NA ND 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 + 18/21 + ALV-J 28/03/02 Albumens 10 C/O NA 10/10 + All negative 04/04/02 Whole blood 120 C/O bc ND 2/120 + 1/60 ALV-A 02/05/02 Serum 60 ND 50/60 + ND ND 08/05/02 Whole blood 40 C/O bc ND ND All negative 08/05/02 Serum 188 ND

ND 81/158 + 14/30 +

ND ND

ND ND

15/05/02 Serum 120 ND ND ND

All negative 26/40 + 11/20 +

ND ND ND

ND ND ND

29/05/02 Albumens 67 C/O NA 27/67 + All negative 29/05/02 Serum 180 ND 63/180 + ND ND 20/06/02 Albumens 63 C/O NA 34/63 + 18/22 neg for

ALV-J 26/06/02 Serum 49 ND

ND All negative

11/20 + ND ND

ND ND

22/07/02 Whole blood 43 C/O bc ND 9/43 + All negative 30/07/02 Meconium 99 C/O NA 1/99 + 1/1 + ALV-J 31/07/02 Serum 30 ND All negative ND ND 22/08/02 Serum 30 ND 4/30 + ND ND 11/09/02 Whole blood

C-swabs Feathers

100 100 100

C/O bc ND ND

93/100 + NA NA

3/100 + ND ND

3/3 + ALV-J ND ND

16/09/02 Serum 30 ND 13/30 + ND ND 20/09/02 Whole blood 50 C/O bc ND 3/50 + 3/3 + ALV-J 20/09/02 Tumours 2 ND NA NA 2/2 + ALV-J/A 02/10/02 Albumens 40 C/O NA All negative ND 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 + 7/24 +

14/16 + ALV-J

22/10/02 Albumens 320 C/O ND 53/320 + All negative 22/10/02 Albumens 67 C/O NA All negative ND 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 ND 4/100 + ND ND 28/11/02 Whole blood

Feathers 48 48

ND ND

All negative NA

ND ND

ND ND

17

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

29 29 29

C/O bc ND ND

2/29 + NA NA

8/29 + ND NA

ND ND ND

09/01/03 Whole blood C-swabs Feathers

12 12 12

C/O bc ND ND

All negative NA NA

All negative ND ND

ND ND ND

09/01/03 Whole blood C-swabs Feathers

13 13 13

C/O bc ND ND

1/13 + NA NA

2/13 + ND ND

2/2 + ALV-J ND

2/2 + ALV-J 09/01/03 Whole blood

C-swabs Feathers

25 25 25

C/O bc ND ND

16/25 + NA NA

All negative ND ND

ND ND ND

21/01/03 Tumours 2 C/O NA 2/2 + ND 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 ND 21/60 + 10/21 + ALV-J 11/02/03 Whole blood 60 C/O bc 13/60 + 14/60 + ND 11/03/03 Albumen 1 C/O NA + Albumen

+ after culture ALV-J + ALV-A negative

19/03/03 Albumen 1 C/O NA + Albumen + after P1

Neg after P2

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 ND 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 ND 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 + 13/90 + 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 + 10/31 + Negative 09/04/03 Whole blood 60 C/O bc

C/O bc 17/30 + 14/30 +

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 + 16/60 + Negative

17/04/03 Whole blood 43 C/O bc

C/O bc 12/22 + 19/21 +

3/22 + 4/21 +

1/3 + ALV-A Negative

30/04/03 Whole blood 60 C/O bc 32/60 + 9/60 + 1/9 + ALV-A

18

Date

Sample type

No. of Samples

Cultured

Ab-ELISA

Ag-ELISA

PCR

30/04/03 Whole blood 65 C/O bc C/O bc

32/32 + 28/30 +

2/32 + 2/33 +

Negative Negative

30/04/03 Whole blood 90 C/O bc All Negative All negative ND 01/05/03 Whole blood 66 C/O bc

C/O bc 11/34 + 28/32 +

3/34 + 4/32 +

Negative 1/4 ALV-A

05/05/03 Whole blood 60 C/O bc C/O bc

21/30 + 25/30 +

1/30 + 2/30+

Negative Negative

15/05/03 Tumours 2 ND NA 2/2 + after culture

1/2 ALV-A

27/05/03 Whole blood 270 C/O s ND All negative ND 28/05/03 Whole blood 191 C/O s

C/O s ND ND

2/63 + All negative

Negative ND

04/06/03 Whole blood 180 C/O s ND 2/180 + Negative 12/06/03 Whole blood 270 C/O s ND 3/270 + 1/3 ALV-A 12/06/03 Whole blood 265 C/O s ND All negative ND 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 ND ND

All negative All negative

ND ND

27/06/03 Whole blood 300 C/O s ND All negative ND 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. ND = not tested. NA = not applicable.

4.12 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-

19

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

16

12

na

10*

1

62.5

Whole blood

3824

3736

238 (6.37)

50

13

1.68

Buffy-coat

1918

1830

216 (11.80)

50

11

3.33

Serum

1906

1906

22 (1.15)

nil

2

0.10

Albumens

1122

1122

151 (13.35)

19

nil

1.69

Meconiums

99

99

1 (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.

20

A.

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40 50 60 70

Samples

positivenegative

0.6

B.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 10 20 30 40 50 60 70

0.6

C.

0

1

2

3

4

5

6

0 10 20 30 40 50 60 70

0.6

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)

21

Table 4.8 ALV-J infection status in various Australian flocks.

Category Flock Genotype Age No tested

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 278 30 (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.

ALV-J isolation ALV-J antibody

Genotype No flocks tested No of flocks

positive No flocks tested No of flocks

positive

A

40

13 (32.2)

34

18 (52.9)

B

2

2 (100.0)

2

2 (100)

C

5

0 (-)

5

0 (-)

D

1

0 (-)

1

0 (-)

Total

48

15 (31.25)

42

20 (47.6)

22

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)

00.5

11.5

22.5

3

0 50 100Sample

SP r

atio

PCR negativeALV-A PCR positive

0.2

gs ELISA (DF-1)

00.5

11.5

22.5

33.5

0 50 100Sample

PCR negativeALV-A PCR positive

0.2

Figure 4.7 C/O vs DF-1 background with ALV-Ag ELISA.

23

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 4.10. 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”.

4.15 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.

24

Table 4.10 Confirmatory results for Australian samples (3.03) forwarded to the AFRC Compton, UK in November 2002.

Sample no.

p27-ELISA IAHL

p27-ELISA Compton

PCR UOM

PCR Compton

1. + - J Negative 2. + + A+J A+J 3. + - J Negative 4. + + A+J A+J 5. + + A+J A+J 6. + + A+J A+J 7. + + A+J A+J 8. + + A+J A+J 9. + + A+J A+J

10. + + A+J A+J 11. + + A+J A+J 12. + + A+J A+J 13. + - A A 14. + - Negative Negative 15. + - Negative Negative 16. + + A A 17. + - Negative Negative 18. + - Negative Negative 19. + - Negative Negative 20. + + A+J A+J 21. + + A+J A+J 22. + + A+J A+J 23. + + A+J A+J 24. + + J J 25. + + J J 26. + + A+J A+J 27. + + A+J A+J 28. + + J J 29. + + J J 30. + + J J

25

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.

26

Table 5.1 Virus micro-neutralisation assay results for various ALV-J isolates.

Virus Strain

Antiserum

HPRS-103

ADOL-Hc1

J98290/

191

UOM-101

UOM-202

UOM-217

UOM-219

ADOL-Hc1

+

+

-

-

-

+

+

J98290/191

+

+

+

-

+

-

-

UOM-101

-

+

+

+

+

-

+

27

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 105 TCID50 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

28

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

Body weight (g) (Mean ± SD) Group

Day old 3 weeks 6 weeks

UOM-201 46.6 ± 3.76 a 687.7 ± 101.79 a 1495.2 ± 274.34 a UOM-224 45.5 ± 4.61a 676.3 ± 110.04 a 1386.5 ± 311.71 a Control 46.2 ± 5.00 a 853.7 ± 100.63 b 1803.5 ± 177.53 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

Bursa weight to body weight ratio x10, 000 (Mean ± SD)

Group 6 weeks 12 weeks 18 weeks

UOM-201 18.65 ± 4.34 a 9.65 ± 3.15 a 5.26 ± 2.74 a UOM-224 21.11 ± 3.24 b 12.78 ± 3.62 b 8.94 ± 3.38 b Control 21.26 ± 3.91 b 12.90 ± 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-201 25.48 ± 5.00 c 19.95 ± 7.94 c 16.14 ± 0.32 b 14.81 ± 12.81 a

UOM-224 19.89 ± 7.82 b 16.51 ± 5.00 b 24.31 ± 2.72 c 13.71 ± 3.37 a

Control 10.08 ± 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

Mortality (no) Age Group A Group B Group C

0-1 weeks 2/35 2/35 0/35 1-3 weeks 1/33 1/33 0/35 3-6 weeks 0/27 0/27 0/30 6-12 weeks 1/22 1/22 1/25 12-18 weeks 6/17 0/17 0/19 18-26 weeks 0/5 0/7 0/8

29

Table 6.5 Infection Status of ALV-J infected birds at different ages

UOM-201 UOM-224 Age 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 105 TCID50 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.

30

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).

31

• 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.

32

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 sub-group 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.

33

LTR LTRgag pro env

envH5 H7b AD1* Env-A

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)

J5’ J3’

2124 bp env region

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

34

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 non-specific 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).

35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

401 bp544 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

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

401 bp544 bp694 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 2. 694bp 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.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

544 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.

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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.

1 2 3 4 5 6 7 8 9 10 11

2124 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 tm37. 1. 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.

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8.3 Cloning and sequencing of the env region The PCR fragments successfully amplified in Figure 8.1 were cloned into the pGEM-T vector using the TA cloning system (Promega). Potential positive clones were screened by PCR with the primers H5/H7b and positive plasmids grown and extracted with QIAprep Spin Miniprep kit (Qiagen) and sequenced using Big Dye Terminator reagents (Applied Biosytems). Sequencing reactions were performed in the IAHL and submitted for electrophoresis to the AGRF (WEHI). The complete sequence of the env region was compiled in six separate sequencing reactions (Figure 8.2), using both M13 forward and reverse universal primers and primers designed toward ALV-J (JF1, JF2, JR1, JR2). The complete sequence was compiled using ANGIS software.

2124 bp env clones

J5’ J3’

M13 rev M13 for JF1 JF2 JR2 JR1

Full sequence of ALV-J env achieved in 6 separate sequencing reactions.

1. 2.

3.

4. 5.

6.

Figure 8.2 Schematic of sequencing strategy.

8.4 Sequence analysis and phylogeny The sampling is narrow and limited to just four clones, thus care should be taken when endeavouring to draw conclusions about the relationship between these isolates and those from the UK and USA. This is particularly difficult when considering the time difference involved. (HPRS was first isolated and sequenced more than ten years prior to our Australian isolates). The sampling is narrow in that the Australian clones sequenced are from the same time period (December 2001) and two of the clones sequenced are from the same source (and not surprisingly these clones share the greatest homology with each other). An attempt was made to clone the env sequence from J98290/191 (Figure 8.1, lane 8) but due to the decreased level of amplification observed for this isolate it failed to clone. Thus it would appear that isolation from a particular source has had the greatest influence on the relationship between the clones.

8.5 Sequence comparisons Sequence comparisons of the Australian isolates with both HPRS-103 (UK) and ADOL Hc1 (USA) are shown in Table 8.2. This is a simple comparison representing % homology between each sequence. The degree of similarity ranges from 89.6% (ADOL-Hc1 and Clone-14) up to the highest homology between the two isolates from the same source at 97.7% (Clone-3 and Clone-4).

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Table 8.1 Identity of sequenced clones.

Clone #

Date of isolation

Sample type

Age

Source.

UOM #

Clone-1

Dec. 2001

Whole blood

Breed A 54 wo

A

UOM-201

Clone-3

Dec. 2001

Whole blood

Breed B 30 wo

B

UOM-216

Clone-4

Dec. 2001

Whole blood

Breed A 54 wo

B

UOM-219

Clone-14

Dec. 2001

Whole blood

Breed B

10 do

C

UOM-224

Note: Clones 3 and 4, although from different breeds (both A and B), have been isolated from the same Source (B).

Table 8.2 Sequence comparisons: Australian isolates to HPRS-103 and ADOL-Hc1.

ADOL-

Hc1

HPRS-103

Clone-1

Clone-3

Clone-4

Clone-14

ADOL-Hc1

100%

94.8%

94.4%

95.6%

94.8%

89.6%

HPRS-103

100%

93.6%

95.5%

95.7%

89.9%

Clone-1

100%

95.0%

93.9%

90.3%

Clone-3

100%

97.7%

92.9%

Clone-4

100%

93.8%

Clone-14

100%

8.6 Phylogenetic analysis of Australian isolates A simple phylogenetic analysis is shown in Figure 8.3. This figure shows clearly that the Australian isolates cluster together and are thus more related to each other than they are to ADOL and HPRS sequences. Interestingly, Clones 3 and 4 that share the highest degree of homology do not cluster with each other. Taken as a whole the Australian isolates are more closely related to HPRS-103 than they are to ADOL-Hc1. This is reflected in the average homology of all four clones being 93.6% when compared to HPRS-103 and 91.1% when compared to ADOL-Hc1. The difference however is marginal and clouded when just breed A or breed B isolates are considered by themselves. In this

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situation breed A isolates and breed B isolates are not distinguishable based on their relationship to HPRS-103 and ADOL-Hc1 sequences.

AADDOOLL--HHcc11

HHPPRRSS--110033

CClloonnee--1144

CClloonnee--44

CClloonnee--11

CClloonnee--33 Figure 8.3 Phylogenetic analysis of Australian isolates.

A representation of the phylogenetic analysis using the complete env (GP85 and TM37) sequences of: ADOL-Hc1 USA prototype strain, HPRS-103 UK prototype strain, Clone-14 Australian isolate from source C/breed B, Clone-4 Australian isolate from source B/breed A, Clone –1 Australian isolate source A,/breed A, Clone-3 Australian isolate source B/breed B. 8.7 Conclusions

Taken as a whole, the Australian isolates of ALV-J that have been sequenced so far are most likely more related to HPRS-103 than they are to ADOL-Hc1. When considering either breed A or breed B origin of the isolates, no definitive relationship can be established. Thus from this limited analysis, the Australian isolates are likely to have originated from the UK and cannot be distinguished definitively as either breed A or breed B. It should be stated that the sampling is limited, as two samples, one from breed A and one from breed B, were isolated from the same source (B). It is impossible to tell if this isolate arrived there via breed A birds and then moved over to breed B birds (or visa versa). It is however unlikely to have arrived in both, as these two clones are the most similar to each other that we have sequenced (i.e. some cross contamination occurred at source B or prior to this).

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9. Development of practical method-ologies for the detection of ALV-J

9.1 Introduction Virus isolation studies carried out during this project identified numerous isolates of ALV-J (and ALV-A) from Australian flocks. The prevalence of the virus however, appears to be low; somewhere in the order of 1-2%. In order to sample the number of birds required to obtain a reasonable degree of confidence at this rate of prevalence a large number of samples is predicted to be necessary. For detection of 1% prevalence with a 95% confidence ~270 samples will need to be screened per flock. The preferred ‘gold standard’ for virus isolation is the use of a 24 well format (5 x 105 cells/well in 1 ml of M199 +1% NBS) with culture for two passages of five days and inoculation from buffy-coat preparations (Appendix G). This method however is relatively labour intensive, expensive and is not conducive to the easy handling of large numbers of samples. The use of a different format for virus isolation is needed to enable the more efficient handling of many samples. In consultation with industry veterinarians and chicken breeding companies, a proposal for the use of less cells in a shortened culture time-frame with an alternative inoculum to buffy-coat preparations has been explored. The exact parameters for the 96 well culture format implemented and a comparison to the 24 well format is described here. 9.2 A 96 well culture format of C/O CEF’s for virus isolation The use of a 96 well format is standard for many serological procedures and multi-channelled pipettors make the handling of large numbers of samples relatively easy. In order to increase the ease of handling of the large number of samples required to reach 95% confidence, with a prevalence of infection at ~1%, a 96 well culture format for virus isolation was proposed. The procedure adopted is described in Appendix D. In brief it involves the inoculation of 50,000 cells per well with 25 ul of serum isolated from clotted whole blood. The cells are incubated for 2 days in 100ul of medium, the medium is then changed (adding 200ul/well) and the cells incubated for a further 2 days when another medium change is performed (again adding 200ul/well). Cells are incubated a further 2-3 days and cultures are then tested by ALV-Ag ELISA. Any positive reactions are then tested by PCR. The essential differences between this approach and the 24 well format used previously is the reduction in the number of C/O CEF’s used per sample and a reduced culture period (7 days vs 2 x 5 days). Cultures are also inoculated with serum rather than buffy-coat preparations. This is envisaged to reduce the level of background false positives observed in buffy-coat inoculations (believed to be caused by the presence of residual RBC’s). Table 4.7 shows that 12% of buffy-coat samples tested by Ag-ELISA are positive (SP ratio >0.2) after 24 well culture, serum inoculations in the 96 well format in comparison show ~1% of samples as positive (SP ratio >0.1). A number of medium changes have been included in this new format for the purpose of removing any contamination that may also increase the level of false positives. These medium changes will, however, lead to a reassessment of the minimum SP ratio used as a cut off for detection of positives (usually 0.20). Experience shows this may have to be lowered to 0.10. A serial diluted positive control viral stock is also included in each isolation to monitor the sensitivity.

9.3 A comparison of 24 well format vs 96 well format An assessment of the sensitivity of the proposed 96 well format in comparison to the 24 well format was carried out. Both ALV-A and ALV-J viral stocks were serially diluted (neat to 10-7) and inoculated into C/O CEF’s in both formats. The inclusion of 20mg/ml DEAE in the tissue culture medium was also investigated as this has been demonstrated to enhance viral infectivity in culture. The results are summarised in Table 9.1. The inclusion of DEAE was able to improve the sensitivity of C/O CEF’s to infection some 10 fold when compared to cells cultured in the 96 well format without DEAE. The direct comparison of the 24 well format with the 96 well format +DEAE showed

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equal levels of detection (both at 10-1 dilution) when an ALV-J viral stock was used. The 24 well format did show a 10 fold increase in sensitivity when compared to the 96 well format +DEAE (10-5 vs 10-4). These results demonstrate an equivalent sensitivity of both culture methods (24 well and 96 well +DEAE) when using an ALV-J viral stock. It is important to note that this experiment does not compare the differences in sensitivity of virus isolation that may occur between using buffy-coat versus serum as inoculums. Preliminary results from using this new 96 well format (summarised in Table 4.7) indicate that very few positive virus isolations have been confirmed using this procedure. Although the level of ALV-Ag positives has been reduced when compared to buffy-coat isolations using the 24 well format. These results may be misleading given that the selection of samples for testing during the early stages of this project were flocks identified as possibly having problems with ALV-J; later submissions including all those used in the 96 well format were tested on the basis of screening for the virus. These flocks were not identified as having any particular problem but are being screened to ensure freedom from ALV-J. This selective bias is likely to have reduced the probability of detecting positive isolations. 9.4 Comparison of 96 and 24 well formats in field samples The previous experimental comparison of the 96 well and 24 wells formats was using a laboratory viral stock. A much more informative comparison would be a direct comparison of positive field samples using both buffy-coat and serum samples in both formats of cell culture. There is, however, some limited published information that indicates virus isolation using buffy-coat maybe more effective than serum. Koch et al. (2000) infected day old chicks with both ALV-A and ALV-J and performed virus isolation from plasma and white blood cells (WBC’s) at 2 weeks old, 13 weeks old and 21 weeks old. For sampling at 21 wo there were 47/96 (49%) isolations from plasma and 74/90 (82%) for WBC’s. This significant difference was not observed at 2 weeks old (plasma 100%, WBC’s 99%) or as marked at 13 weeks old (plasma 47%, WBC’s 66%). There are a number of other differences in the methodology used from those described here; for example the use of serum versus plasma and the method of WBC’s preparation is not explained in this paper. A comparison of serum vs buffy-coat in both 24 and 96 well format is currently underway. 9.5 What Samples are recommended? Sampling needs to be as simple as possible but must ensure the best possibility of achieving successful virus isolation. Successful isolation from tumours, meconium, albumen, whole blood and feathers have been demonstrated. Each of these samples has its advantages and disadvantages. Tumours are likely to arise at later ages and are thus unsuitable for early detection of ALV-J. Meconium is useful in that it can be easily taken from young chickens without causing unnecessary stress. However, there is always the possibility of birds being non-shedders and these birds can potentially evade detection. Albumen is another good source of viable virus for isolation but is only available after lay. The advantages and disadvantages of direct detection from PCR of feathers has been discussed earlier (Chapter 5). A degree of variability and questionable sensitivity lead to the non-recommendation of this procedure for reduction/eradication. Whole blood is a relatively easy sample to obtain and is easily processed in the laboratory to obtain serum or buffy-coat and it can also be screened for ALV-J antibody. Figure 4.6 depicts the SP ratio profiles from antibody positive flocks. If the percentage of positive birds is >40 % with reasonable SP ratio levels (>2.0) and the flock is 20 weeks old then the ALV-J Ab-ELISA is a useful tool for identifying flocks that should be further investigated by virus isolation. Due to its versatility and reliability, it is recommended to use whole blood as the primary sample for virus isolation and detection of ALV-J. It should be stressed that other samples described here can be extremely valuable and can in some circumstances give a rapid definitive diagnosis of ALV-J and thus one sample type should not be relied upon solely or any overlooked as useful.

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Table 9.1 Sensitivity for detection of ALV-J and ALV-A in C/O CEF culture in 96 well format (+/- DEAE) versus 24 well format.

J J J A A A Dilution -DEAE 96 +DEAE 96 24 well -DEAE 96 +DEAE 96 24 well

Neat 1.82 1.96 2.34 2.32 3.30 3.51 2.39 2.74 1.95 1.79 3.33 3.69 10-1 0.02 0.01 1.76 1.58 3.28 3.09 2.27 2.57 2.54 2.41 3.50 3.39 10-2 0.01 0.01 0.02 0.10 0.00 0.01 1.57 2.58 2.28 2.36 3.32 3.28 10-3 0.01 0.01 0.01 0.02 0.01 0.00 0.89 2.23 2.47 2.09 3.36 3.41 10-4 0.02 0.02 0.02 0.02 0.01 0.02 0.19 0.03 0.62 1.26 3.30 3.25 10-5 0.02 0.02 0.01 0.01 0.01 0.02 0.01 0.03 0.01 0.01 3.28 3.16 10-6 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.03 0.02 0.01 0.00 0.00 10-7 0.02 0.03 0.02 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.02

Table shows SP ratio of gs ELISA (positives >0.2 are shaded and bolded; other potential positives are bolded only). J = stock of Australian isolate J98. A = stock of RSV-A. -DEAE 96 = M199 media/50,000 cells per well in 96 well format (seven day culture x media @day 2 and 5). +DEAE 96 = M199 media + DEAE (20mg/ml)/50,000 cells per well in 96 well format (seven day culture x media @day 2 and 5). 24 well = 1ml M199 media/500,000 cells per well in 24 well format (x 2 five day passage/100ul inoculation of passage 1 to 2).

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10. Implications and recommendations The serological, virological and molecular biological procedures for the isolation, detection and characterisation of ALV-J have been established in the International Avian Health Laboratory at the University of Melbourne and are available to the Australian poultry industry. In this project, no attempt has been made to determine the exact sensitivity of the ALV-J PCR assay in terms of the ability to detect limiting target DNA copy numbers. The primary objective was to establish a molecular based diagnostic technique for detecting ALV-J in flocks of chickens rather than in individual birds. In the initial design and characterisation of the H5/H7 primer pair (Smith et al., 1998) this kind of analysis was performed and like many PCR based assays was found to be very sensitive. In addition, the propagation of viral isolates in C/O CEF’s leads to easily detectable levels of proviral DNA in these cells and thus negates the need for an assay that detects to extremely low copy numbers. Thus, characterisation of the sensitivity of various cell lines to propagation of ALV-J was considered a more important undertaking. However, the parameters and conditions for the PCR assay (Appendix F) were established to specifically amplify ALV-J sequences in a sensitive assay and a serially diluted ALV-J proviral control stock was always used to check the sensitivity of each individual assay against these original conditions, which have remained unaltered through the course of this project. The procedures and techniques established during this project have been routinely used to isolate and detect ALV-J from field samples taken in the Australian poultry industry. These procedures have clearly established the presence of ALV-J in Australia (Table 4.6). The antigenic characterisation of Australian isolates by micro-neutralisation assays have revealed that these isolates are antigenically distinct. Sequence data shows distinct nucleotide changes in the env gene and a degree of variability from the original prototypes of ALV-J, to indicate that the Australian isolates also cluster phylogenetically into a distinct group. This implies the possibility of a period of selective isolation. However, the lack of any sequence data from recent overseas isolates makes a definitive conclusion about how recently these isolates of ALV-J entered the Australian industry impossible. The question thus remains - is ALV-J continuing to circulate in isolation in the local industry or is it also being introduced periodically from outside Australia from other sources? The most direct way to answer this question and to ensure that ALV-J is not present in new imports of poultry into this country is to screen these imports upon entry into Australia. During the course of this project only one batch of imported birds was made available to be screened for the presence of ALV’s. Of the 120 samples taken from the same imported batch, one was confirmed to contain ALV-A. The presence of ALV-A in imported stocks suggests that ALV-J may well have entered Australia by the same route. The use of a micro-culture format with the capacity to screen large numbers of samples and reach the level of confidence required for potentially very low levels of prevalence is desirable. The 96 well format culture procedure described in Chapter 9 (and Appendix D) fulfils many of these requirements. This system should be able to meet all the practical requirements for ALV detection, screening, isolation and PCR detection of ALV-J in broiler breeder flocks by the Australian poultry industry. It includes relatively simple sample handling, procedures and virus isolation in C/O CEF’s which will propagate all sub-groups of ALV and thus not limit the scope of potential isolates (the culture period has also been reduced to 7 days to make the process more rapid). Culture is followed by a screen with p27-ELISA, again picking up all sub-groups of ALV but also quickly eliminating samples free of ALV’s, thus effectively reducing the number of samples required to be further tested by the PCR assay. An initial preliminary test of this procedure has been undertaken. An important question remains to be investigated and that is whether serum alone is as effective as buffy-coat preparations for virus isolations. Buffy-coat contains peripheral blood monocytes (PBMC’s), the presence of which may enhance virus isolation in culture. This question remains unanswered As a somewhat unexpected complication to the present project work directed at ALV-J detection, ALV-A has also been isolated from the Australian poultry industry during this study, both on its own and in co-isolations with ALV-J. Little is known about the impact of co-infections of ALV-A with

45

ALV-J on flock health and pathology. This situation does, however, present a greater potential for virus recombination to occur and therefore increase the risk of new ALV viral strains emerging. The presence of solitary isolates of ALV-A from the Australian industry, although widely thought to be of little threat to the broiler industry, is still quite undesirable. The major breeding companies are endeavouring to eliminate all replicative viruses from their elite stocks, as any replicative virus represents a potential economic threat. The circulation currently in Australia of both ALV-J and ALV-A sub-groups therefore presents a potential for a recombinative threat to the Australian poultry industry. Positive steps will need to be taken to detect and reduce this threat both locally and by overseas suppliers of broiler breeding stocks in the future. The completion of the present project has yielded ALV-J detection technology of considerable precision (using a combination of serology, virology and molecular biology). Conclusive evidence of the distribution of ALV-J infections in breeder flocks in several states of Australia has also been obtained during these studies, 2000-2003. ALV-J is egg-transmitted and a tumour-causing pathogen of broiler breeders which is known to be capable of causing significant levels of economic loss. A logical development using the outputs from this present project would be for the Australian chicken meat industry to engage in an active program of ALV-J reduction in its breeding stocks. This approach should comprise two phases, these being, firstly the detailed checking of all future imported broiler stocks to ensure ALV-J freedom and, secondly, quarantine measures on local flocks found to be infected with ALV-J so that these contaminated lines can be ‘washed’ out of the industry and replaced as soon as may be practicable.

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11. References Arshad, S.S., L.M. Smith, K. Howes, P.H. Russell, K. Venugopal and L.N. Payne (1999) Tropism of subgroup J avian leukosis virus as detected by in situ hybridisation. Avian Pathology 28: 163-169. Bagust, T.J. (1993) Reticuloendotheiosis Virus p437-454 Chapter in Virus Infections of Birds Ed J.B. McFerran and M.S. McNulty, Elsevier, Amsterdam. Bagust, T.J., Whithear, K., White, N.J., O'Rourke, D. and Mitchell, M. (1999) Scoping studies on infectious causes of immunodepression and transmissible viral proventriculitis in Australian broiler flocks. Report to RIRDC of Project UM43A RIRDC Publication p1-27. Bagust, T.J., Reddy, M.R. and Fenton, S.P. (2002). Avian leukosis virus subgroup-J (ALV-J): Molecular technologies to understand virus evolution and eradication programs. Proceedings of 7th WPSA Asian Pacific Federation Conference, 6-10 October 2002, Gold Coast Australia. Pp 479-483. Bai, J., L.N. Payne, and M.A. Skinner (1995a) Sequence of host-range determinants in the env gene of a full-length, infectious proviral clone of exogenous avian leukosis virus HPRS-103 confirms that it represents a new subgroup (designated J). Journal of General Virology 76: 181-187. Bai, J., L.N. Payne, and M.A. Skinner (1995b) HPRS-103 (Exogenous Avian Leukosis Virus, Subgroup J) Has an env Gene Related to Those of Endogenous Elements EAV-O and E51 and an E Element Found Previously Only in Sarcoma Viruses. Journal of Virology 69: 779-784. Benson, S.J., B.L .Ruis, A.L. Garbers, A.M. Fadly and K.F. Conklin (1998) Independent isolates of the emerging subgroup J avian leukosis virus derive from a common ancestor. Journal of Virology 72: 10301-10304. Biggs, P.M. (1997) Lymphoproliferative disease of turkeys p 485-488 in Poultry Diseases 10th Edition B. Calnek Ed Iowa State University Press, Ames Iowa, USA. Cavanagh, D. (1993) Advances in avian diagnostic technology. Proceedings of Xth World Veterinary Association Congress; Sydney, pp 57-70. Cavanagh, D., K. Maeditt, K. Shaw, P. Britton and C. Naylor (1997) Towards the routine application of nucleic acid technology for avian disease diagnosis. Acta Veterinaria Hungarica 45: 281-298. Davidson, I., Borenshtain, R. (2002) The feather tips of commercial chickens are a favourable source f DNA for amplification of Marek’s disease virus and avian leukosis virus, subgroup J. Avian Pathology 31 237-240.

Du, Y. and Cui, Z. (2002). Study on the pathogenecity of Chinese strains of subgroup J avian leucosis viruses. Agricultural sciences in china. 1: 586-588. Du, Y., Cui, Z., Qin, A., Silva, R.F and Lee L.F (2000). Isolation of subgroup J avian leucosis viruses and their partial sequence comparison. Chinese Journal of Virology. 16: 342-346. Fadly, A.M. and E.J. Smith (1997) An overview of subgroup J-like avian leukosis virus infection in broiler breeder flocks in the United States p. 54-57. In Proceedings of the Avian Tumor Viruses Symposium. American Association of Avian Pathologists, Kennett Square, Pa. Fadly, A.M. and R.L. Witter (1998) Oncornaviruses: Leukosis/Sarcomas and Reticuloendotheliosis pp 185-196 in A Laboratory Manual for the Isolation and Identification of Avian Pathogens 4th edition Ed. Swayne, D.E. The American Association of Avian Pathologists. Kennet Square PA.

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Fadly, A.M. and E.J. Smith (1999) Isolation and some characteristics of a subgroup J-like avian leukosis virus associated with myeloid leukosis in meat-type chickens in the United States Avian Diseases 43: 391-400. Fadly, A.M., Silva, R.F. and Lee, L.F. (2000) Antigenic characteristics of selected field isolates of subgroup J avian leukosis virus. Proceedings of International symposium of ALV-J and other avian retroviruses. Rauischholzhausen, Germany, pp 13-22. Fadly, A. M., and Payne, L.N. (2003) Leukosis/Sarcoma Group p 465-516 in Poultry Diseases 11th Edition Y. M. Saif Ed Iowa State University Press, Ames Iowa, USA Fadly, A. M. (1998) Subgroup J avian leukosis virus - infection in meat-type chickens: a review. Fourth Asia Pacific Poultry Health Conference, AVPA, Melbourne. Scientific Proceedings p109-112. Fenton, S.P., Reddy, M.R. and Bagust T.J. (2002) Advances in detection of ALV-J in Australia. Proceedings of Australian Veterinary Poultry Association Scientific Meeting, 21-22 November, 2002, Melbourne, Australia. Pp 23-24. Gavora, J.S., J.L. Spencer,. and J. Chambers. (1982) Performance of meat-type chickens test-positive and -negative for lymphoid leukosis virus infection. Avian Pathol 11:29-38. Gavora, J.S., J.L. Spencer, R.S. Gowe and D.L. Harris (1980). Lymphoid leukosis virus infection: effects on production and mortality and consequences in selection for high egg production. Poultry Science 59: 2165-2178. Gingerich, E., Porter, R.E., Lupiani, B. and Fadly, A.M. (2002) Diagnosis of Myeloid Leukosis induced by a recombinant Avian Leukosis Virus in commercial White Leghorn egg-laying flocks. Avian Diseases 46: 745-748. Goodwin, M (1999) Broilers from parents that have subgroup J avian leukosis/sarcoma virus tumors are not as economical to produce as broilers from parents that do not have tumors. Proceedings of the 48thWesten Poultry Disease Conference p96. Hudson, B.P., Wilson, J.L., Zavala, G and Sander, J.E. (2002) Fertility and sperm quality of broiler breeder males infected with subgroup J avian leukosis virus. Avian Diseases. 46:1033-1037. Hwang, C.S and Wang C.H. (2002) Serological profiles of chickens infected with subgroup J Avian Leukosis Virus. Avian Diseases 46: 598-604. Ignjatovic, J. and T.J. Bagust (1983) Practical application of ELISA for detection of vertical transmission of leukosis virus in commercial layer hens. Avian Pathology 12: 515-519. Ignjatovic, J., and T.J. Bagust (1982) Detection of avian leukosis virus with the ELISA system: evaluation of conjugation methodology and comparison of sensitivity with the phenotypic mixing test in commercial layer flocks. Avian Pathology 11: 579-591. Jurajda, V., Kulíková, L., Halouzka, R., Geryk, J., and Svoboda, J. (2000) Avian Leukosis Virus Type J (ALV--J) in the Czech Republic. Acta Veterinaria Brunensis 69: 143-145. Koch, G., Van der Velde, J., Hartog, L., Gielkens, A.L.J. and Landman, W.J.M. (2000) Horizontal and vertical transmission of ALV-J and ALV-A virus in broiler breeders chickens. Proceedings of International Symposium on ALV-J and other Avian Retroviruses (pp. 141-151). Rauischholzhausen, Germany.

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Lupiani, B., Hunt, H., Silva, R. and Fadly, A. (2000) Identification and characterization of recombinant subgroup J avian leukosis viruses (ALV) expressing subgroup A ALV envelope. Virology 276: 37-43. McKay, J.C. (1998). A poultry breeders approach to avian neoplasia. Avian Pathology 27: Suppl. S74-S77. Payne, L.N., Brown, S.R., Bumstead, N. and Howes, K. (1991) A novel subgroup of exogenous avian leukosis virus in chickens. Journal of General Virology 72: 801-807. Payne, L.N. (1998) HPRS-103: a retrovirus strikes back. The emergence of subgroup J avian leukosis virus. Avian Pathology 27: Supplement 1, S36-S45. Payne, LN, Howes, K., Gillespie, A,M, and Smith, L.M. (1992) Host range of Rous sarcoma virus pseudotype RSV(HPRS-103) in 12 avian species: support for a new avian retrovirus envelope subgroup, designated J. Journal of General Virology 73: 2995-2997. Silva, R.F., Fadly, A.M. and Hunt, H.D (2000). Hypervariability in the envelope genes of subgroup J avian leukosis viruses obtained from different farms in the United States. Virology 272: 106-111. Smith, E.J. A.M. Fadly and L.B. Crittenden. (1990). Interactions between endogenous virus loci in ev6 and ev21. Immune response to exogenous avian leukosis virus infection. Poultry Science 69: 1244-1250. Smith, E.J., S.M. Williams and A.M. Fadly (1998). Detection of avian leukosis virus subgroup J using the polymerase chain reaction. Avian Diseases 42: 375-380. Smith, L.M., S.R. Brown, K. Howes, S. McLeod, S.S. Arshad, G.S. Barron, K. Venugopal, J.C. McKay, and L.N. Payne (1998) Development and application of polymerase chain reaction tests for the detection of subgroup J avian leukosis virus. Virus Research 54: 87-98. Spencer, J.L., Chan, M., Nadin-Davis, S. and Chambers, J.R. (1999) Relationship between egg size and ALV-J in eggs from broiler breeders presented at International Conference and Exhibition on Veterinary Poultry, Proceedings, Beijing Editor Yan Hanping p11-17. Spencer, J.L. (1999) Avian leukosis virus subgroup J - prospects for control. Proceedings of the 48th Western Poultry Disease Conference p 99-101. Stedman, N.L., and T.P. Brown (1999). Body weight suppression in broilers naturally infected with avian leukosis virus subgroup J. Avian Diseases 43: 604-610. Stedman, N.L., Brown, T.P. and Brown, C.C. (2001). Localization of avian leukosis virus subgroup J in naturally infected chickens by RNA in situ hybridisation. Veterinary Pathology, 38: 649-656. Sung, H.W., Reddy, S.M. and Fadly, A.M. (2002) High virus titer in feather pulp of chickens infected with subgroup J avian leukosis virus. Avian Diseases, 46: 281-286. Tripathy, D.N. (1998) Use of molecular biology techniques in diagnosis of poultry diseases. Proceedings of 47th Western Poultry Disease Conference, Sacramento, California, pp3-5. Van der Sluis, W. (1998) 1998 world poultry disease update. World Poultry 14: 38-39.

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Venugopal, K., L.M. Smith, K. Howes and L.N. Payne (1998) Antigenic variants of J subgroup avian leukosis virus: sequence analysis reveals multiple changes in the env gene. Journal of General Virology 79: 757-766. Venugopal, K., Howes, K., Flannery, D.M.J. and Payne, L.N. (2000) Subgroup J avian leukosis virus infection in turkeys: induction of rapid onset tumours by acutely transforming virus strain 966. Avian pathology. 29: 319-325. Witter, R.L., Bacon, L.D., Hunt, H.D., Silva, R.E. and Fadly, A.M. (2000) Avian leukosis virus subgroup J infection profiles in broiler breeder chickens: association with virus transmission to progeny. Avian Diseases, 44: 913-931. Witter, R.L. and Fadly, A.M. (2001) Reduction of horizontal transmission of avian leukosis virus subgroup J in broiler breeder chickens hatched and reared in small groups. Avian Pathol. 30:641-654. Zavala, G. (1998) A new challenge for the poultry industry. Myeloid Leukosis. Vineland Update No. 61 April p1-4.

Zavala, G., Jackwood, M.W., and Hilt, D.A. (2002) Polymerase chain reaction for detection of avian leukosis virus subgroup -J in feather pulp. Avian Diseases. 46: 971-978.

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12. 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 (C/O CEF culture and feathers).

Appendix I. Preparation of C/O CEF’s.

Appendix J. DNA molecular weight markers.

Appendix K. The 24 well culture procedure.

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Appendix A: Sample preparation and handling

The following describes the procedures used for the preparation and handling of all samples used in this project. All samples for virus isolation were propagated in the 24 well format described in Appendix K with the exception of serum samples which were inoculated into the 96 well culture format (Appendix D)

Albumen: should be sampled from freshly laid eggs as aseptically as possible, placing ~ 1ml of albumen into a sterile tube. 50 ul of albumen is used for virus isolation by placing it directly into the 24 well culture dish on top of C/O CEF’s that have begun to adhere (wells contain 5 x 105 cells in 1 ml of medium with antibiotics). Whole blood: the handling and preparation of buffy-coat and separation of serum are described in Appendix G. Briefly the buffy-coat fraction is removed after centrifugation of whole blood containing an anti-coagulant (Na citrate) the buffy-coat is present between the plasma and cellular pellet and is drawn off with a glass Pasteur pipet and placed into a clean sterile tube prior to freezing and thawing. 50 ul of buffy-coat preparation is placed on top of C/O CEF’s that have begun to adhere (wells contain 5 x 105 cells in 1 ml of medium with antibiotics). Serum is separated by allowing the whole blood to clot in a sterile tube at room temperature for 1-2 hours (temperatures above 27oC are recommended, lower temperatures will require longer clotting times). Clotted whole blood is then spun briefly and the serum drawn off into a clean sterile tube. 25 ul of serum is used to inoculate cells in the 96 well format, in which the medium contains antibiotic. Tumours: are excised as aseptically as possible and placed in a sterile tube and stored at either 4oC or frozen. Small portions of tumour are ground in a mortar and pestle with a small volume of tissue culture medium. A small volume of ground tissue is used to inoculate cells in the 24 well format on top of C/O CEF’s that have begun to adhere (wells contain 5 x 105 cells in 1 ml of medium with antibiotics). If tumour tissue causes excessive cellular toxicity the medium can be changed several hours after the initial inoculation. Approximately 50 mg of tumour tissue is sufficient for DNA extraction by the method described in Appendix H. Meconium: swabs of meconium and the cloaca are made using a sterile cotton bud and placed in 1 ml of sterile tissue culture medium containing antibiotic to prevent bacterial growth. This tissue culture medium can be used to inoculate cells directly in the 24 well culture format. Extra antibiotics are recommended when isolating virus from meconium and C-swabs (such as gentamycin). Feather: two to three larger feather are plucked and placed in individual bags. New feathers containing moist pulp can readily squeezed and the pulp collected into a sterile clean tube. DNA extraction is described in Appendix H.

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Appendix B: Precautions to avoid PCR contamination

The ability of the polymerase Chain Reaction to amplify a single molecule means that trace amounts of DNA contaminants could serve as templates, resulting in amplification of the wrong template (false positives).

Setting up the laboratory to avoid contamination

The following points should be considered to avoid PCR contamination from sources such as:

- Laboratory benches, equipment, and pipetting devices, which can be contaminated by previous DNA preparations, by plasmid DNA, or by purified restriction fragments.

- Cross contamination between samples.

- Products from previous PCR amplifications (this is likely to be the predominant problem in a

laboratory that routinely carries out PCR detection of the same pathogens).

Laboratory facilities

At a minimum, set up physically separated working places for:

o Template preparation before PCR o Setting up PCR reactions o Post PCR analysis

- Use thin –walled PCR tubes which are DNase and RNase free - Use aerosol-resistant pipette tips, and a dedicated (used only for PCR) set of pipettes, preferably

positive displacement pipettes. - If possible, set up PCR reactions in a dedicated area with UV light sterilisation. Store a microcentrifuge and gloves that are used only for PCR in this area.

- Use sterile technique and always wear fresh gloves when working in the PCR area. Change gloves frequently, especially if you suspect they have become contaminated with solutions containing template.

- Always use new and/or sterilised glassware, plasticware, and pipettes to prepare PCR reagents. - Have your own set of PCR reagents and solutions that are used only for PCR. Store these all reagents in small aliquots to limit contamination should it occur.

- When pippetting DNA, take care to avoid creating aerosols that could carry contaminants. - Always include control reactions, for example a negative (no DNA) control which contains all reaction components except the template DNA, and a positive control that has been successfully used in previous PCRs.

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Appendix C: Collection of blood for ALV monitoring

Collection and transport of samples can be critical for the successful isolation of viable virus For this reason, care should be taken to ensure the best possible sample arrives at the IAHL in the best possible condition. Materials and methods Prior to bleeding it is helpful to notify the IAHL of the expected time of arrival as well as the number of your samples. The bleeding crew should set up in advance with blood collection tubes (1.5 ml eppendorf centrifuge tubes or an equivalent) and syringes. Sharps container to dispose of used syringes/needles. Pre-opening wrapper of the syringes can save time in the shed. Store in zip lock plastic bags with needle ends all going in the same direction in case needle guard falls off. Chickens are to be sampled by collecting blood from the wing vein using a 3-5 ml syringe with a 20G one inch needle in an aseptic manner. One ml of blood will be deposited into the tubes (collect a little more than needed in the syringe ~1.5ml). Prevent any “bubbles" in the tubes and try to keep the same volume level in all tubes. Also practice caution to prevent overfilling or rapid filling that will increase cell lyse as well as the possibility of splash contamination. The samples should not be allowed to drop below 27oC during the collection process or serum separation will be a problem (Samples can be held at the birds body temperature without deterioration during the collection process as long as the serum is left in contact with the blood clot). Samples should remain at room temperature for as long as it takes for serum/blood separation to occur (normally one to two hours), more time may be necessary in cool temperatures. The samples should be cooled down to ~4oC when clear serum is visible in the blood sample tube. Samples should not be held in a location such as direct sunlight or high heat. The samples should be transported on ice as soon as possible to the IAHL UOM (hopefully within 24 hrs of collection). They should at no stage be stored on site for shipment at a later stage. If samples can not be sent directly the serum may be pulled off into tissue culture 96 well sterile plates or sterile tubes in an aseptic manner and then frozen at –80oC using dry ice. They must then remain at –80oC/on dry ice for shipment (shipment on dry ice is costly and most couriers will be reluctant to perform this service. Thus this approach is not recommended). Notifying the IAHL of your bleeding schedule and number of samples in advance will assist the laboratory in preparing adequate cell culture material/preparation to run the assay. This can be done by Fax to 03 83449676.

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Appendix D: C/O CEF micro-culture for monitoring of ALV’s

Materials Blood to have been collected using a 3-5ml syringe with a 20G needle (one per bird via the wing vein) carefully placing 1ml into 1.5ml eppendorf tubes. Sampling at x270 samples per flock. Sample processing Upon arrival of 1ml of whole blood in 1.5 ml eppendorf tubes place the tubes in the bench top centrifuge (Rotor holds x24 samples) and centrifuged at 10,000 rpm for 2 mins. This allows complete separation of the serum that is then carefully drawn off the clot (~200ul) with a pipetor (clean sterile tip for each sample) and placed in a sterile 96 well tissue culture plate (90 samples per plate with the remain six left empty). This plate is then replicated by drawing 25 ul from each well and placing it into another sterile 96 well plate. The remaining original ~175 ul is then frozen/stored in the –70oC freezer. The 25 ul replica plates is then cultured immediately with C/O CEF’s (or if unavoidable also stored at –70oC until cultured). Preparation of 1% M-199 M-199 medium is usually purchased at 10x concentration To make 500ml:

- 50ml 10x M-199 media (MultiCel, with Hanks salts) - 5 ml L-Glutamine (Gibco, 200mM) - 5ml bicarbonate (7.5%) - 5ml 100x Antibiotic (Gibco) - 5ml NBS (Gibco) - 500ul DEAE mixture (20mg/ml filter sterilised) - H20 to 500ml (429ml) Age for 24 hours before use Store at 4oC Warm to 37oC before use

Virus control preparation

Initially an RSV-A cell free viral stock is prepared and stored in 100ul aliquots at –70oC (x96 capped cluster tubes). After freezing an aliquot is thawed and serially diluted 10-1 to 10-8 in chicken serum and plated out in 96 well format with 25ul from each dilution with 50,000 C/O CEF’s per well and 100ul 1% M-199. A 200ul medium change (1% M-199) is performed at day 2 and day 5 with a gs ELISA performed on day 7 after freezing and thawing (assay 100ul of media). The cut off point of detection is determined. All subsequent virus isolations use these 100ul frozen RSV-A control aliquots as positive controls. The stock is serially diluted in chicken serum and three dilution points are inoculated at 25ul per well. The dilution points are either side of the point of lowest detection (i.e. detection cut off is 10-4 then include 10-3, 10-4, 10-5 as controls on each plate (wells 91, 92 and 93) the remaining wells (94, 95 and 96) are negative controls (containing cells plus 25ul of clean chicken serum)

Micro-culture procedure

Serum from samples is removed as soon as possible after arrival and 200 ul is placed in 96 well plate. 25 ul is removed immediately and placed in another 96 well sterile culture plate. The remaining 175 ul of serum is frozen at –70oC for backup if required. 270 samples is recommend per flock for ALV-J monitoring this means x90 samples are added to each plate. The remaining 6 wells are for positive and negative controls. See Figure 1 for plate layout for storage, culture and ELISA. Control viral

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RSV-A aliquot is thawed and serially diluted in chicken serum (Gibco), by placing 10 ul of this stock into 90 ul of chicken serum. Diluted the stock out to 10-5 and place 25 ul of the 10-3,10-4 and 10-

5 dilutions into wells 91, 92, and 93. 25 ul of normal serum is added to wells 94, 95 and 96. An ampoule of 1.8 x107 C/O CEF’s is thawed and placed into 30 ml of 1%M199 +DEAE and 100 ul aliquots of cells (50,000) are placed into each well with a multi-pipettor, being careful not to cross contaminate wells. Cells are placed at 37oC with 5% CO2. After two days incubation the medium is drawn off and replaced with 200 ul of fresh warm medium. A further two day incubation under the same conditions prior to another medium change with 200 ul of warm fresh medium is the final step prior to ALV-Ag ELISA monitoring. The medium changes are carried out using a multi-pipettor to remove the old medium (a new tip for every well) again ensuring there is no cross contamination. The cell layer which is confluent at the point of the first medium change is monitored at each point to ensure the cells remain attached. While in the incubator plates are placed in zip lock plastic bags to ensure no cross contamination with other different flocks occurs and to reduce evaporation from the plates. The ELISA is performed 2-3 days after the final medium change, after the cells have been frozen and thawed. 100 ul of the medium is used for the ELISA test. The 10-4 positive viral dilution is used on the ELISA as a tissue culture positive control to ensure the virus isolation process has worked efficiently, one of the uninfected wells is used on the ELISA as a tissue culture negative. The 4 remain wells on the 96 well ELISA plate are for 2 positive and two negative ELISA controls used for the calculation of the SP ratio. Any positive samples detected by ELISA are harvested for DNA extraction (Appendix H) and PCR (Appendix F) and repassaged to confirm positivity. Under this procedure any SP ratio upon ELISA that is greater than 0.10 is considered positive and should be subjected to PCR for ALV-J and ALV-A (Appendix F) and repassaged to confirm the original finding. This lowering of the cut off point for positives is necessary because of the number of medium changes and the relatively short time period (2 days) that cells remain in the final medium change prior to the ELISA being performed. In the 24 well culture format this final incubation is 5 days allowing an increase in the levels of p27 present in the culture well.

Repassaging positive samples

Any positive samples from the original virus isolation are passaged for a second time in C/O CEF’s. The remaining ~ 100 ul of the first passage is removed from the well and the cells harvested for DNA extraction as described in Appendix H. The medium supernatant remaining after cells have been removed is used to inoculated the second passage. 10 ul of this medium is added to 25 ul of chicken serum and added to a 96 well plate usually setting x 5 replicates of each positively identified sample. C/O CEF’s are added in the same concentration and procedure as for the first passage (including medium changes and ELISA monitoring after culture). Any positives are again checked by PCR. Results A sample is considered positive only after conformation by PCR with the H5/H7b primers. A positive ALV-Ag ELISA does not confirm the presence of ALV-J. Positive ELISA results after virus isolation can be result from a number of different possibilities:

1. The presence of ALV-J in the sample (confirmed by H5/H7b PCR) 2. The presence of ALV-A in the sample (confirmed by H5/env-A PCR) 3. The presence of ALV-E in the sample (can be eliminated by passaging through DF-1

cells) 4. The presence of another ALV sub-group (B, C, or D) these sub-groups are unlikely to be

present in chicken samples but can be eliminated by the use of the primers H5/AD1 which will amplify all sub-groups of ALV’s (having already eliminated the involvement of ALV-J, A and E a positive amplification with these primers would point to the involvement of B, C or D).

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A.

ABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12

All blank

1 9 17

8 16 24

89

90

32 40 48 56 64 72 80 88

25 33 41 49 57 65 73 81

B.

A B C D E F G H

1 2 3 4 5 6 7 8 9 10 11 12

10-3

10-4

10-5

-ve

-ve

-ve

Positive Controls

Negative Controls

C.

ABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12

-ve

10-4

-ve

-ve

Tissue CultureControls

IDEXControls

+ve

+ve

Figure 1.

A. 96 well layout for serum samples for storage B. 96 well layout for C/O CEF culture. C. 96 well layout for subsequent ELISA screening.

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Appendix E: Interpretation and calculations for ALV-Ag and ALV-J Antibody test

ALV-Ag ELISA (IDEXX)

For this assay to be valid, the difference of absorbance (A) at 650nm between the Positive control and the Negative control (PC-NC) should be greater than 0.200. The negative control mean absorbance should be less than or equal to 0.150. The presence or absence of p27 antigen is determined by relating the A(650) value of the unknown to the positive control mean. The positive control mean has been standardised and represents significant antigen levels (approximately 10 ng/ml). The relative level of antigen in the unknown can be determined by calculating the sample to positive (SP) ratio. If this ratio is less than or equal to 0.2, the sample should be considered negative. SP ratios greater than 0.2 indicates the presence of p27 antigen. Calculations Negative control mean (NC) = well A(650) + well A(650) 2 Positive control mean (PC) = well A(650) + well A(650) 2 SP ratio (SP) = Sample mean –NC PC-NC

ALV-J Antibody ELISA (IDEXX)

For this assay to be valid the difference of absorbance (A) at 650nm between the positive control mean and the negative control mean (PC-NC) should be greater than 0.10. In addition, the negative control mean absorbance should be less than or equal to 0.150. For invalid tests, technique may be suspect and the assay should be repeated. The presence of antibody to ALV-J is determined by relating the A(650) value of the unknown to the positive control mean. The positive control has been standardised and represents significant antibody levels to ALV-J in chicken serum. The relative level of antibody in the unknown can be determined by calculating the sample to positive SP ratio. If this ratio is less than or equal to 0.6, the sample should be considered negative. SP ratios greater than 0.6 indicate the presence of antibody to ALV-J. Interpretation of results

1. The ALV-J antibody test has been developed as a flock screening tool for monitoring horizontal transmission of the virus. The ALV-J status of individual birds cannot be assessed.

2. ALV-J seroconversion is variable across lines and may depend on endogenous leukosis virus expression. Testing of meat type birds less than 12-14 weeks of age is not recommended.

3. A positive result on the ALV-J antibody test kit indicates exposure to the ALV-J virus; antibody titer does not indicate whether the virus is being actively shed. A determination of ALV-J flock status should include testing for the virus.

4. Vertical transmission of ALV-J results in seronegative, immune tolerant progeny.

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Calculations Negative control mean (NC) = well A(650) + well A(650) 2 Positive control mean (PC) = well A(650) + well A(650) 2 SP ratio (SP) = Sample mean –NC PC-NC

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Appendix F: ALV-J PCR conditions and procedure

Primers: All primers used in the IAHL at the University of Melbourne are ordered from Invitrogen life technologies. The primers arrive lyophilised and can be stored in the freezer until working dilutions are made. A x10 stock solution is made according to the synthesis report accompanying the oligonucleotide order. This stock is made to 200 pmol/ul and stored in small aliquots (100 ul) at –20oC. The stock is diluted 1:10 prior to use.

PCR master mix The total number of sample to be tested is used to calculate the volumes used for the PCR master mix (remember to include all the necessary control, PCR +/-, tissue culture +/-) and allow +10% for overage.

Example: 20 samples (+ 4 controls + 3 overage)

Make master mix for 27 samples Master mix (ALV-J): x1 x27 10x PCR buffer 2.5 ul 67.5 ul Mg Cl2 (50mM) 1.0 ul 27 ul dNTP Mix (40mM each) 0.25 ul 6.75 ul Taq (2 Units/ul) 0.5 ul 13.5 ul Primers (H5 and H7b) 2 ul 27 ul of each Template 5 ul 5 ul from each sample Milli-Q 13.75 271.5 ul

Total 25 ul 20/tube + template

Add 20 ul of the master mix to each tube (which have been clearly labelled) and then add template to each tube. Include a positive control in this for ALV-J a HPRS-103 proviral DNA extract that has undergone a 5 fold serial dilution to determine the detection cut off. The dilution point prior to this detection cut off is used as the PCR positive control (a similar RSV-A DNA stock is used as a positive for ALV-A). PCR tubes are briefly spun in the centrifuge prior to loading into the PCR machine.

The following conditions have been established to specifically amplify ALV-J:

1. 95oC 4 mins 35 cycles of: 2. 95oC 30 sec 3. 58oC 30sec (for ALV-A H5/env-A 55oC) 4. 72oC 30 sec 5. 72oC 10 mins 6. Hold at 4oC

Products are visualised by running 1.5% (w/v) 1 x TPE agarose gels with DNA molecular weight markers (1 kb ladder Appendix J). ALV-J positive gives 544 bp fragment. ALV-A positive gives a 694 bp fragment. Note: If a negative result for ALV-J PCR is obtained the primers Baf and Bar can be used to monitor DNA extraction. These primers recognise the chicken β-actin gene and a positive result indicates successful extraction of DNA from C/O CEF’s.

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Appendix G: Buffy-coat preparation. This procedure describes the preparation of a crude buffy-coat preparation that is suitable for virus isolation in a 24 well format (Appendix K) The use of this crude buffy-coat preparation is unsuitable for using in the 96 well format as toxicity becomes a problem. Materials - Sterile 1.5 ml eppendorf tubes - Whole blood with anti coagulant (Na Citrate) Method 2-5 ml of whole blood is placed in an appropriate tube with an anti-coagulant (3.5% Na citrate). Ensure thorough mixing of blood and anticoagulant to prevent blood clotting. The whole blood is then centrifuged (generally in 1.5 ml eppendorf tubes) for 5 min at 3-4,000 rpm. Centrifugation causes separation of the plasma and cells. The plasma can be drawn off and frozen separately for antibody testing or virus isolation should this be necessary. A glass pasture pipette is used to carefully remove the buffy-coat. The buffy-coat is the layer of cells at the interface between the plasma and the major cellular pellet. This buffy-coat preparation is placed in a clean sterile 1.5 ml eppendorf tube then frozen and thawed. 50 ul of this preparation is used for virus isolation in 24 well format (Appendix K)

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Appendix H: Genomic DNA extraction from C/O CF’s and feathers

Genomic DNA can be extracted by numerous methods and a number of commercial kits are available for this purpose. These kits can be expensive when dealing with a large number of samples. This protocol describes a method that is relatively simple and inexpensive. This procedure primarily describes the extraction of DNA from C/O CEF’s cultured in a 24 well format (4-5 x 105 cells/well in 1ml of 1% M199 medium). However, it is also applicable to genomic extraction from tumours or tissue samples as well as other cell types in 24 or 96 well format. Where appropriate alterations to the protocol will be indicated. Materials - Sterile 1.5 ml eppendorf tubes - Bench top microcentrifuge - Sterile 200 ul pipet tips Reagents: - Lyses buffer (4 M Guanidine HCl, 25mM Na Citrate, 1% Triton X-100) - Phenol/chloroform/isoamylalcohol. - 100% Isopropanol - 70% Isopropanol

- Feather lysis buffer (0.5% SDS, 0.1M NaCI, 10mM Tris (pH 8.0), 1mM EDTA) Method After virus isolation C/O CEF’s are generally frozen and thawed (x2) prior to ALV-Ag ELISA of the tissue culture medium. If cultured samples have been frozen/thawed the cells will have detached from the culture dish and will need to be harvested from the medium. The ~1ml of the media is placed in 1.5 ml eppendorf tube (making sure to transfer as many cells as possible). Cells are pelleted by centrifugation for 2 min at 10,000 rpm. Tissue culture media is removed and stored at –70oC and can be used to inoculate further passages of isolated virus should this be necessary. The pelleted cells are dissolved in 200 ul of lyses buffer. For feathers feather tip 3-4 mM is placed in a sterile eppendorf tube (or the pulp can be squeezed out into the tube) with 100 ul of feather lysis buffer and incubated at 55oC for 2-4 hrs. For cells in 96 well format 100 ul of lyses buffer is sufficient (200 ul is also sufficient buffer to solubilise ~50 mg of tissue). If cell culture has not been previously frozen and thawed then media can be removed ensuring the cell layer remains attached and 200 ul of lyses buffer can be added directly to the culture plate. Samples are incubated at room temperature for 5 mins with vortexing until the cells have solublised. The lyses buffer (feather lyses buffer) is then extracted x2 with an equal volume of phenol/chloroform/isoamylalcohol (25:24:1) with vigorous vortexing and separation of phases by centrifugation for 5 min at 10,000 rpm. After the second extraction the upper clear aqueous phase is removed into a clean sterile 1.5 ml eppendorf tube (~180 ul) and the DNA is precipitated with an equal volume of 100% isopropanol by mixing completely and incubating at room temperature for 15 mins. DNA is pelleted by centrifugation for 15 mins at 14,000 rpm. Isopropanol is removed being careful not to dislodge the pellet (which may or may not be visible). The pellet is washed with ~200 ul of 70% isopropanol and centrifuge at 14,000 rpm for 5 mins prior to air drying at room temperature. The pellet is resuspended in 20-50 ul of sterile Milli-Q H20 and stored at 4oC prior to PCR.

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Appendix I: Preparation of C/O CEF’s Materials 9-11 day old C/O embryonated eggs. (dead/infertile eggs are discarded)

- Sterile scissors - Sterile forceps - Sterile filter mesh - Sterile flask - Sterile magnetic flea - 10 ml centrifuge tubes - Sterile petri dishes - Haemocytometer - Sterile pipettes - 70% Ethanol Reagents PBS (Ca-Mg free) - 0.25% Trypsin, warmed to 37oC - Trypan blue stain (Gibco 0.4% in PBS) - Growth medium (M199 + 5% NBS) Method To begin ensure the biohazard or laminar flow cabinet is clean and sterile (wipe with 70% Ethanol). The entire procedure will be carried out in the biohazard hood. - Swab eggs with 70% Ethanol and allow to dry. - Using sterile instruments open eggs above the air sac and remove the embryo and place into sterile

petri dish (note: Six embryo’s will yield approx. 1 litre of CEF cells) - Remove and discard the head and feet of all embryos - Rinse x3 with PBS (discarding PBS after each rinse) - Mince the embryo tissue with scissors (or force the tissue through a 10ml syringe without a needle)

into a sterile flask containing a magnetic flea. - Rinse the minced tissue three times with PBS (discarding PBS after rinse). - Add approx. 20 ml of warm Trypsin and stir on magnetic stirrer for 5 mins. - Decant the Trypsin supernatant fluid and discard. - Add a further 40 ml of Trypsin and stir for 10 mins. - Decant trypsin supernatant into a bottle containing 10 ml of NBS (Trypsin inactivation). - Repeat Trypsinisation step until all tissue is processed (2-3 times) - The entire pool of trypsinised cells is now filtered through filter mesh (fine and coarse) and placed

into centrifuge tubes and centrifuged for 5 mins at 1000 rpm. - Supernatant is discarded and cells resuspended in 10 ml of growth medium. - Perform a viable cell count using 1:10 and 1:100 dilutions in trypan blue. - After quantitation seed the cells at the following rates:

- 4-4.5 x 105 cells/ml for confluency in one day - 3x 105 cells/ml for confluency in two days

- Make up the required dilution of cells with growth media. - Dispense cells into culture flasks/plates and incubate at 37oC with 5%CO2.

Freezing of C/O CEF’s:

- Prepare CEF cells as above and grow until confluent. - Remove medium from cultures and wash x 2 with PBS. - Cover monolayer with Trypsin EDTA solution and incubate at 37oC to allow the monolayer to detach. - Observe the monolayer for signs of detachment (the cell sheet detaching in small flakes or a granular appearance as the cells separate from each other). - Once cells have detached, add a quantity of growth medium and pipette up and down until a homogenous single cell suspension is obtained.

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- Perform viable cell count and freeze cells at the appropriate density: o Usually 2 x 107 cells/ml o For 96 well format 1.8 x 107 cells are frozen in 1 ampoule in 1.5 ml.

(upon thawing this is enough cells to seed 3 x 96 well plates at 50,000 cells per well with ~ 20% excess for to allow for cellular death).

o For 24 well plates 1.8 ml of cells at 2 x 107 cells/ml. This allows enough cells to seed 3 x 24 well plates at 5 x 105/cells per well.

- Cells are frozen in M199 medium + 10% NBS and 10% DMSO. - Dispense appropriate volume and density of cells into cryogenic ampoules. - Freeze cells by placing them in a styrofoam rack in an esky at –70oC overnight (the aim is to freeze

cells slowly to prevent excessive loss of viability) next day transfer frozen ampoule to liquid nitrogen.

Thawing frozen C/O CEF’s

- Remove ampoule from liquid nitrogen and place directly into 37oC water. - Monitor ampoule in warm water until contents have completely thawed. - Transfer contents of the ampoule to an appropriate volume of 37oC M199 medium +1% NBS.

Seeding 96 well plates (x3) volume used is 30 ml (100 ul/well). For 24 well plates (x3) add cells to 75 ml of medium (1 ml/well).

- Aliquot the appropriate volume into sterile culture plates. Note: For 96 well format M199 medium should contain 20 mg/ml DEAE to enhance viral infection. 96 well plate will also contain 25 ul of sample serum prior to adding the cells. Trypsin solution (for CEF isolation) 2.5 % Trypsin (Gibco, 2.5%) 10 ml (0.25%) Antibiotic/mycotic (Gibco, 100x) 1 ml PBS (Oxoid, Ca-Mg free) to make 100 ml. Trypsin EDTA solution (for freezing CEF’s) 2.5 % Trypsin (Gibco, 2.5%) 4ml (0.1 %) 2% EDTA (Gibco BRL) 1ml (0.02%) Antibiotic/mycotic (Gibco, 100x) 1 ml PBS (Oxoid, Ca-Mg free) to make 100 ml M199 medium 10x M199 stock (MultiCel, 10x) 50 ml Antibiotic/mycotic (Gibco, 100x) 5 ml Sodium bicarbonate (7.5%) 5 ml L-Glutamine (MultiCel, 200mM) 5 ml Milli-Q water to make 500 ml Maintenance Medium 1x M199 + 1% New born serum (Gibco) Growth Medium 1xM199 + 5% New born serum (Gibco)

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7.5% Sodium Bicarbonate Sodium Hydrogen Carbonate (BDH) 7.5g Milli-Q water to make 100 Filter solution through 0.22 um filter.

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Appendix J: DNA molecular weight markers.

250 bp

500 bp

750 bp1000 bp

1500 bp2000 bp2500 bp3000 bp4000 bp5000 bp6000 bp8000 bp10,000bp

All PCR gels depicted in this report have been run using a 1kb ladder as the DNA molecular weight markers. The position of each band in this ladder is indicated above. 1 kb DNA ladder from Promega (cat # G5711). Is used for determining the size of double stranded DNA from 250 to 10,000 base pairs. The ladder consists of 13 blunt-end fragments ranging in length from 250 to 10,000bp in evenly-spaced increments. Easy identification of the 100bp and 300bp fragments is ensured with the increased intensity of these bands

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Appendix K: The 24 well culture procedure. Virus isolation was primarily performed in a 24 well culture format. The number of cells and volume of culture medium enables the isolation of virus from buffy-coat, cloacal swab, tumour tissue and albumen with out excessive cellular toxicity.

Materials - three 24 well culture plates - sterile 10 ml pipettes - 100 ml sterile bottle Reagents - Frozen ampoule of C/O CEF’s (1.8 ml of 2 x 107 cells/ml) - 75 ml of 37oC 1% M199 medium. - Virus inoculum (buffy-coat, albumen etc.) Method An ampoule of C/O CEF’s (1.8 ml of 2 x107 cells/ml) are thawed rapidly after removal from liquid nitrogen and placed in 75 ml of 1% M199 and plated out into three 24 well tissue culture plates at 1 ml/well. Cells are monitored for cellular adherence. Once cells had begun to round up and adhere to the culture plate (~ 1 hour) the viral inoculum was added. This was 50 ul of frozen and thawed buffy-coat preparation from whole blood samples (Appendix G) or 50 ul of albumen sample or 100 ul of tissue culture medium from a meconium or cloacal swab. Cells were incubated for 5 days at 37oC in 5% CO2 with daily monitoring, if excessive toxicity occurred medium was changed to dilute to toxic effects of the inoculum. After the initial 5 days of culture the cells are frozen and thawed (x2) and 100 ul of medium used to inoculate an identical second passage that is again incubated at 37oC for a further 5 days. After freeze thawing (x2) 100 ul of tissue culture medium is screened by ALV-Ag ELISA. Any positives are then subject to DNA extraction (Appendix H) and PCR with ALV-J specific primers (Appendix F).

avian leukosis virus sub-group j (alv-j) leukosis virus sub-group j (alv-j) developing laboratory...

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