Development and Application of Reverse Transcriptase Nested ...

AVIAN DISEASES 47:41–53, 2003

Development and Application of Reverse Transcriptase Nested Polymerase Chain Reaction Test for the Detection of Exogenous Avian Leukosis Virus Maricarmen Garcı´a,A John El-Attrache,A Sylva M. Riblet,A Vagner R. Lunge,BC Andre´ S. K. Fonseca,C Pedro Villegas,A and Nilo IkutaBC Department of Avian Medicine, Poultry Diagnostic and Research Center, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-4875 B Universidade Luterana do Brasil, Canoas, Rio Grande do Sul, Brazil C Simbios Biotecnologia, Canoas, Rio Grande do Sul, Brazil

A

Received 11 December 2002 SUMMARY. A polymerase chain reaction (PCR) assay that utilizes nested primers to amplify a fragment of the long terminal repeat of exogenous avian leukosis virus (ALV) was developed and evaluated for detection of ALV subgroup J directly from clinical samples. Compilation of sequence data from different endogenous and exogenous ALVs allowed the selection of a conserved set of nested primers specific for the amplification of exogenous ALV subgroups A, B, C, D, and J and excluded amplification of endogenous viruses or endogenous viral sequences within the chicken genome. The nested primers were successfully used in both PCR and reverse transcriptase (RT)-PCR assays to detect genetically diverse ALV-J field isolates. Detection limits of ALV-J isolate ADOL-Hc1 DNA by nested PCR and RNA by RT–nested PCR were superior to detection of group-specific antigen by enzyme-linked immunosorbent assay (ELISA) in cell culture. Detection of ALV-J in cloacal swabs by RT– nested PCR was compared with direct detection by antigen-capture (ac)-ELISA; RT–nested PCR detected fewer positive samples than ac-ELISA, suggesting that RT–nested PCR excluded detection of endogenous virus in clinical samples. Detection of ALV-J in plasma samples by RT–nested PCR was compared with virus isolation in C/E chicken embryo fibroblasts; the level of agreement between both assays as applied to plasma samples ranged from low to moderate. The main disagreement between both assays was observed for a group of plasma samples found positive by RT–nested PCR and negative by virus isolation, suggesting that RT–nested PCR detected ALV-J genome in plasma samples of transiently or intermittently infected birds. ALV-J transient and intermittent infection profiles are characterized by inconsistent virus isolation responses throughout the life of a naturally infected flock. RESUMEN. Desarrollo y aplicacio´n de la prueba de la transcriptasa reversa/reaccio´n anidada en cadena por la polimerasa para la deteccio´n de virus exo´genos de la leucosis aviar. Se desarrollo´ y evaluo´ una prueba de reaccio´n en cadena por la polimerasa que emplea iniciadores anidados para amplificar un fragmento de las terminacio´n repetida larga del virus exo´geno de la leucosis aviar, para la deteccio´n directa del virus de la leucosis aviar subgrupo J a partir de muestras clı´nicas. Mediante la compilacio´n de datos de secuencias de diferentes virus endo´genos y exo´genos de la leucosis aviar, se logro´ seleccionar un grupo de iniciadores anidados conservados especı´ficos para la amplificacio´n de los virus exo´genos de la leucosis aviar subgrupos A, B, C, D y J. Dichos iniciadores no amplificaron virus endo´genos ni secuencias virales endo´genas localizadas dentro del genoma del pollo. Los iniciadores anidados fueron empleados exitosamente tanto en la prueba de reaccio´n en cadena por la polimerasa como en la transcriptasa reversa/reaccio´n en cadena por la polimerasa, para detectar los aislamientos de campo gene´ticamente diferentes del virus de la leucosis aviar subgrupo J. Los lı´mites en la deteccio´n del ADN del aislamiento ADOL-Hc1 del virus de la leucosis aviar subgrupo J mediante la prueba de la reaccio´n anidada en cadena por la polimerasa y del ARN mediante la prueba de transcriptasa reversa/reaccio´n anidada en cadena por la polimerasa fueron superiores a la deteccio´n del antı´geno especı´fico de grupo mediante la prueba de inmunoensayo con enzimas asociadas en cultivo celular. Se comparo´ la deteccio´n del virus

41

42

M. Garcı´a et al.

de la leucosis aviar subgrupo J a partir de hisopos cloacales mediante la prueba de transcriptasa reversa/reaccio´n anidada en cadena por la polimerasa con la prueba de inmunoensayo con enzimas asociadas con captura de antı´geno. La prueba de transcriptasa reversa/reaccio´n anidada en cadena por la polimerasa detecto´ un nu´mero menor de muestras positivas que la prueba de inmunoensayo con enzimas asociadas con captura de antı´geno, sugiriendo que la prueba de la transcriptasa reversa/reaccio´n anidada en cadena por la polimerasa no detecto´ los virus endo´genos en las muestras clı´nicas. La deteccio´n del virus de la leucosis aviar subgrupo J en las muestras de plasma mediante la prueba de transcriptasa reversa/reaccio´n anidada en cadena por la polimerasa fue comparable con el aislamiento del virus en fibroblastos de embriones de pollo C/E. El nivel de concordancia entre las dos pruebas, aplicadas a las muestras de plasma, fue de baja a moderada. El principal desacuerdo entre las dos pruebas se observo´ en el grupo de muestras de plasma positivas a la prueba de transcriptasa reversa/ reaccio´n anidada en cadena por la polimerasa y negativas por aislamiento del virus, lo cual sugiere que la prueba de la transcriptasa reversa/reaccio´n anidada en cadena por la polimerasa detecto´ el genoma del virus de la leucosis aviar subgrupo J en las muestras de plasma de aves infectadas de forma intermitente o´ transitoria. Los perfiles de la infeccio´n intermitente o´ transitoria por el virus de la leucosis aviar subgrupo J se caracterizan por las respuestas inconsistentes al aislamiento viral durante la vida de los lotes infectados naturalmente. Key words: RT–nested PCR, virus isolation, avian leukosis virus subgroup J, ac-ELISA Abbreviations: ac 5 antigen capture; ADOL 5 Avian Disease and Oncology Laboratory; ALV 5 avian leukosis virus; CEF 5 chicken embryo fibroblast; dNTP 5 deoxynucleoside triphosphate; DTT 5 dithio threitol; EDTA 5 ethylenediaminetetraacetic acid; ELISA 5 enzyme-linked inmmunosorbent assay; env 5 envelope; FN 5 false negative; FP 5 false positive; gs 5 group-specific antigen; LTR 5 long terminal repeat; M-MLV 5 Moloney murine leukemia virus; PCR 5 polymerase chain reaction; RAV 5 Rous associated virus; RFLP 5 restriction fragment length polymorphism; RSV 5 Rous sarcoma virus; RT 5 reverse transcriptase; SE 5 sensitivity; SP 5 specificity; S/P 5 sample to positive control absorbance ratio; TCID50 5 mean tissue culture infective dose; TN 5 true negative; TP 5 true positive; VI 5 virus isolation

Exogenous avian leukosis viruses (ALVs) are type C retroviruses associated with a variety of neoplasms including lymphoid and myeloid leukosis. Unlike endogenous viral genes that are inherited as host genes, spreading of exogenous ALVs in commercial poultry is through vertical and horizontal transmission (6). These viruses are prevalent in the poultry industry worldwide and cause severe economic losses from tumor mortality, condemnations, and loss of pedigree birds. ALVs infecting chickens can be classified into subgroups on the basis of the specific envelope surface glycoprotein responsible for specific viral interference patterns, virus neutralizing antibodies, and host range (18). Six subgroups of ALVs have been identified that infect commercial poultry (A, B, C, D, E, and J). Subgroups A and B are the most common ALVs in commercial poultry (4). Subgroups C and D have been rarely reported in commercial poultry (18). Subgroup E viruses include the ubiquitous endogenous leukosis viruses of low pathogenicity (22). Subgroup J, a recently discovered member of the leukosis/sarcoma group,

was first isolated from meat-type chickens (17,20). ALV-J behaves as an exogenous virus causing mainly myeloid leukosis and nephromas in meat-type chickens (10,15,19). ALV-J infection can affect meat-type birds at all breeding generations as well as commercial broiler flocks (10). Myeloid leukosis induced by ALVJ emerged in the 1990s as a serious cause of mortality and other production problems in meat-type chickens throughout the world. Current methods for detection of ALVs in chickens include detection of viral group-specific antigen (gs) p27 by the antigen-capture (ac) enzymelinked immunosorbent assay (ELISA) test (25). Monoclonal antibodies against ALV have been developed and are also being used in testing samples for the presence of ALV p27 (3,9). This test is not specific for exogenous virus and will detect gs antigens of endogenous virus in serum, plasma, albumen, and meconium. Because p27 is shared by endogenous and exogenous ALVs, direct ELISA assays cannot be used in identification and differentiation of ALVs. In order to increase the specificity of the assay,

RT nested PCR exogenous avian leukosis virus

propagation of the virus in different phenotypes of chicken embryo fibroblasts (CEFs) is required prior to the ac-ELISA test. The phenotypes of CEFs used most commonly to isolate and detect ALV-J include C/O, which are susceptible to infection from all exogenous and endogenous ALV subgroups, and C/E, which are susceptible to infection only from exogenous ALV subgroups (7,9,11). Virus isolation and identification in cell culture is quite readily used by the primary breeder industry and is often referred to as the ‘‘gold standard’’ for ALV isolation and identification. A more effective control of ALV infections mainly depends on the early detection and removal of infected birds to reduce contact with infected birds and the incidence of horizontal spread (24,29). For almost a decade, a major concern of the primary breeder and associated industries has been to use applicable diagnostic assays to detect and identify ALV-J (17,27). The institution of multiple stringent ALV-J screening procedures, which in the laboratory environment often involves virus isolation on C/E cells coupled with ac-ELISA along with more extensive screening and testing, has reduced the prevalence of ALV-J in the primary breeder industry (14,16). Preparation of primary and secondary C/E CEFs for ALV-J detection and screening requires a minimal 2 days of preparation and at least seven additional days to obtain a result (9). In addition, extensive laboratory materials are required and the procedures utilized are quite labor intensive. Therefore the use of molecular techniques for better diagnostic assistance and ALV-J eradication from breeder flocks has been suggested (13,14,16,23,24). ALV-J–specific polymerase chain reaction (PCR) methods have been developed to detect proviral DNA from buffy coats, infected CEFs (23), and directly from serum, whole blood (24), and feather tips (8,24). Most of the primers used for PCR assays have been developed from sequences of the envelope (env) or long terminal repeat (LTR) regions of the ALV genome (23,24). One set of primers specific for ALV-J amplification has been developed by Smith et al. (23); these are located on the 39 noncoding region of the genome, one primer in the E element and the other primer in the U5 region (23). Another common set of primers utilized in diagnostics is the H5-H7 located at the 39 region of the polymerase gene

43

and the 59 region of the gp85 portion of the env gene (24). These assays have proven to be specific and sensitive for the detection of ALVJ. However, the ongoing mutation trend of the ALV-J env gene (21,26,28) and the increasing number of deletions around the E element region of the ALV-J genome (12) justified examination of other areas of the genome for the amplification of ALV-J. The objective of this study was to develop a reverse transcriptase (RT)–nested PCR assay able to specifically amplify exogenous ALVs and to compare this assay with conventional assays for detection of ALVs. The application of this assay in the detection of ALV-J directly from clinical material such as plasma and cloacal samples and comparison with virus isolation and ac-ELISA are discussed. MATERIALS AND METHODS Viruses, chicken lines, and tumor samples. ALV subgroup A strains RPL-42, RPL-40, and RAV1; subgroup B strain RAV-2; subgroup C strain RAV49; subgroup D strain RAV-50; subgroup E strain RAV-60; and subgroup J strains ADOL-Hc1, ADOL-7375, ADOL-4817, ADOL-7341, ADOL5369, ADOL-5569, ADOL-7501, ADOL-5701, ADOL-6803, and ADOL-7818 were obtained from the U.S. Department of Agriculture, Avian Disease and Oncology Laboratory (ADOL), East Lansing, MI. ALV-J strain HPRS-103 was obtained from the Institute of Animal Health, Compton, Berkshire, United Kingdom. All ALV isolates were propagated in CEFs susceptible to all subgroups following procedures as previously described (11). Whole blood from nine congenic and noninbred chicken lines were obtained from ADOL. Tumor samples from birds were obtained from several commercial broiler chicken operations throughout Brazil and the southeastern United States. Plasma samples. Four sets of plasma samples were collected from three different grandparent flocks and from one pedigree flock. A total of 298 samples were collected from grandparent flocks; 100 samples from flock A (37 wk of age), 100 samples from flock B (26 wk of age), and 98 samples from flock C (16 wk of age). A total of 86 samples were collected from pedigree flock D (40 wk of age). Whole blood was individually collected in 5-ml-draw ethylenediaminetetraacetic acid (EDTA) Vacutainert tubes (Becton Dickinson, Franklin Lakes, NJ). Plasma samples were separated from whole blood samples after centrifugation of the Vacutainert tube at 4 C for 10 min at 1000 3 g. Each plasma sample was divided in two separate tubes and frozen at 280 C until used for virus isolation and RT–nested PCR.

44

M. Garcı´a et al.

Table 1. Nested LTR primers for avian leukosis virus amplification. Primers Leu3.2F Leu7RA Leu11FC Leu12RC

A

LTR

Primer position

U3 U5 U3 U5

7514–7543 7813–7834 7594–7617 7785–7806

Sequence B

GGA ATC CGT TCA

AAT CGC CGA GGG

GTA TTC TTG AAT

GTG ATG GTG CGA

TTA CAG GAA CGG

TRC GTG GTA TCC

RAT ACT CTT ATG CTC AGG TGG GGC C

External nested primers. Primer position as compared with ALV-J strain HPRS-103 (GenBank accession number Z46390). C Internal nested primers. A B

Viral nucleic acid isolation. DNA was extracted from CEF monolayers of ALV subgroups A, B, C, D, E, and J, from tumor samples, and from whole blood of different chicken lines. Depending on the sample source, modifications of the same basic procedure were utilized to extract DNA. Briefly, samples were incubated for 1 hr at 65 C with 100 ml of lysis buffer (5% sodium dodecyl sulfate in 0.05 M EDTA) and 100 mg of proteinase K (Amresco, Solon, OH). After incubation, DNA was extracted with phenol : chloroform : isoamyl alcohol (25:24:1) followed by a chloroform extraction (1:1) and precipitated with isopropanol in the presence of 3 M sodium acetate for 1 hr at 220 C. DNA was pelleted by centrifugation at 14,000 rpm (Eppendorf 5417R; Brinkmann Instruments, Westbury, NY) for 20 min. Pelleted DNA was rinsed with 70% ethanol and resuspended in 30 ml of double-distilled water for use in subsequent PCR amplification reactions. RNA was extracted from cell-free CEF supernatants, from cloacal swab suspensions, and from plasma samples by a modification of the method previously described by Chomzcynski and Sacchi (5). Briefly, 100 ml of tissue culture supernatant, cloacal sample suspensions, or plasma sample was added to 400 ml of complete D solution. Samples were mixed gently and incubated for 15 min at 60 C. After incubation, 400 ml of phenol, 80 ml of chloroform, and 40 ml of 3 M sodium acetate were added. To separate the aqueous phase from the phenol phase, samples were gently mixed and centrifuged at 12,000 g for 15 min. Once the aqueous phase was separated, 500 ml of cold isopropanol was added and the sample was incubated for 15 min at 280 C. After RNA precipitation, the sample was centrifuged fro 10 min, the supernatant was removed, and the RNA pellet was washed with ethanol. RNA pellets were resuspended in 20 ml of double-distilled water with 0.25 ml of 100 mM dithiothreitol (DTT) and 0.2 ml of 10 U/ ml RNAse inhibitor (Life Technologies, Rockville, MD). Sequence analysis. The 39 LTRs of different ALV-J isolates and from ALV-J–induced tumor samples were amplified and sequenced with primers located at the DR1 and U5 noncoding regions of the

HPRS-103 strain genome (2). PCR products were obtained for isolates ADOL-Hc1, ADOL-7375, ADOL-4817, ADOL-7341, ADOL-5369, and ADOL-5569 and from tumor samples TM1, TM2, TM3, TM4, TM5, TM6, and TM7. Amplification products were sequenced at the Molecular Genetics Instrumentation Facility, The University of Georgia, Athens, GA. Sequences were aligned with Megalign program (DNASTAR, Madison, WI) with the Clustal alignment algorithm. The aligned sequences were formatted for phylogenetic analysis by the maximum parsimony method with 1000 bootstrap replicates in a heuristic search with PAUP 3.1 software (Sinauer Associates, Inc., Champaign, IL). The nucleotide sequence data obtained for seven ALV-J isolates and seven tumor samples have been deposited in GenBank Data Libraries. The assigned accession numbers are AF448392 (TM1), AF448393 (TM2), AF448394 (TM3), AF448395 (TM4), AF448396 (TM5), AF448397 (TM6), AF448398 (TM7), AF448399 (ADOL-4817), AF448400 (ADOL7375), AF448401 (ADOL-7341), AF448402 (ADOL-5369), AF448403 (ADOL-5569), AF448404 (ADOL-7501), and AF452249 (ADOLHc1). LTR primers specificity. One set of external primers (Leu3.2F/Leu7R) and one set of internal primers (Leu11F/Leu12R) were designed (Table 1). The specificity of external and internal primers was tested for different strains of exogenous ALVs, subgroups A, B, C, D, E, and J, and nine inbred and congenic chicken lines with known endogenous gene loci (1) (Table 2). All amplification reactions were performed in 50 ml volume in a PTC-200 thermocycler (MJ Research, Inc., Watertown, MA). The reaction mixture contained 5 mM of each primer, 1 mM deoxynucleoside triphosphates (dNTPs), 0.4 ml of 5 U/ml Taq polymerase (Promega, Madison, WI), 1 ml of 103 Taq buffer, and 0.8 ml of 25 mM MgCl2. Template concentrations from infected CEFs and chicken blood ranged from 20 to 50 ng of DNA. The cycling parameters included an initial denaturation step of 4 min at 94 C followed by 30 cycles of denaturing at 94 C for 20 sec, annealing at 60 C for 40 sec, and extension at 72 C for 60 sec. The final

RT nested PCR exogenous avian leukosis virus

Table 2. Specificity of LTR primers. Chicken lines and ALVs 7.6-VB*S1 (1,2)C 0 (none)C 15B1 (1)C N (1,3,6)C P (?)C EV21 (21)C ALV-6 (ALV-A env)C C (1,7,10)C 72 (1,2)C 71 (1,2)C 15I5 (1,6,10)C 63 (1,3)C RAV-1 (A)D RPL-40 (A)D RPL-42 (A)D RAV-2 (B)D RAV-49 (C)D RAV 50 (D)D RAV-60 (E)D ALV-JE

LTR primers ExternalA InternalB Nested 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 2 1

2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 2 1

2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 2 1

A Leu3.2F/Leu7R—amplification product sizes ranged from 350 to 318. B Leu11F/Leu12R—amplification product sizes ranged from 175 to 220. C Endogenous gene loci in parentheses. D ALV subgroups in parentheses. E ALV-J isolates—ADOL-Hcl, 7375, 4817, 7341, 5369, 5569, 7501, 5701, 6803, 7818, and IAHHPRS-103.

cycle was followed by an extension step of 72 C for 5 min. Nested PCR. As with single PCR reactions, the specificity of the nested primer reaction was tested with DNA from different strains of exogenous ALV subgroups A, B, C, D, and J, endogenous virus subgroup E, and DNA from nine congenic and inbred chicken lines with known endogenous genes loci (1) (Table 2). The nested PCR was assembled in a single tube format by adding the internal primer reaction mixture into the tube where the external primer amplification was performed. The first amplification was assembled in a 10 ml reaction volume. The first amplification had 1.0 ml of 1 mM dNTPs, 1.0 ml of 0.5 mM Leu3.2F, 1.0 ml 0.5 mM Leu7R, 0.4 ml of 5 U/ ml Taq polymerase (Promega), 1.0 ml of 103 Taq buffer, and 0.8 ml of 25 mM MgCl2. Template concentrations from infected CEFs and chicken blood ranged from 20 to 50 ng of DNA. The second amplification was assembled in a 40-ml reaction volume. Different from the first amplification, the second am-

45

plification reaction mixture contained the internal primers at a 5 mM concentration with no template added as required for a nested amplification reaction. The first amplification of the nested reaction was carried out for 15 cycles of denaturing at 94 C for 20 sec, annealing at 60 C for 40 sec, and extension at 72 C for 60 sec. The second amplification of the nested reaction was carried out for 35 cycles at the same temperature and time parameters as the first amplification. RT. In order to amplify viral RNA from cell-free CEF supernatants, from cloacal swab suspensions, and from plasma samples, a RT reaction was performed prior to nested PCR. The RT reaction was performed in a 10-ml reaction volume. The RT reaction was assembled with 2.0 ml of 200 U/ml Moloney murine leukemia virus (M-MLV) RT M-MLV (Life Technologies), 2.0 ml of 53 M-MLV buffer, 1.2 ml of 1 mM dNTPs, 0.25 ml of 100 mM DTT, 0.5 ml of 5 mM Leu7F primer, and 0.2 ml of 10 U/ml RNase inhibitor. RNA template concentrations of 100–200 ng extracted from cell-free CEF supernatants were utilized. For RT reactions on cloacal and plasma samples, 2.0 ml of template was used consistently. The reaction was incubated for 1 hr at 37 C followed by 2 min at 94 C. After the RT reaction, the nested amplification was assembled in one tube as described above by adding 10 ml of the external primer reaction mixture. The second amplification reaction mixture contained the same components as described above but was assembled in a final volume of 30 ml. The amplification parameters used for RT– nested PCR were the same as the parameters used for nested PCR amplifications. Restriction enzyme analysis. Restriction enzyme analysis of the 39 LTR sequences was performed by MapDraw program (DNASTAR). Amplification products obtained with the nested PCR were digested with DdeI and TaqI restriction enzymes (NewEngland BioLabs Inc., Beverly, MA) following the manufacturer’s recommendations. Digestion reactions were subjected to electrophoresis on a 12.5% polyacrylamide gel (Invitrogen, Carlsbad, CA) and visualized by rapid silver staining (Pharmacia, Biotech, Inc., Piscataway, NJ). Detection of ALV-J Hc1 strain by ac-ELISA, nested PCR, and RT–nested PCR. Six 10-fold dilutions of ADOL-Hc1 isolate at a titer of approximately 104.5 mean tissue culture infective dose (TCID50) ml were prepared in maintenance medium and simultaneously inoculated on secondary C/E CEFs (Kestrel, Inc., Waukee, IA). Each viral dilution was inoculated in replicas of five and incubated overnight; afterward, the medium was replaced by standard maintenance medium and cells were held in the incubator for 7 days. After incubation, one of the plates was processed for p27 ac-ELISA detection. Briefly, 40 ml of a 5% Tween 80 was added per well

46

M. Garcı´a et al.

Fig. 1. Relationship of U3 LTR sequences among ALVs, RSV, and endogenous viral (ev) genes. The phylogram was generated by the parsimony analysis in heuristic search and neighbor joining group method with branch distances. Sequences marked with asterisk (∗) were generated in this study. Published sequences have GenBank accession number in parentheses. Sequences designated as TM1 to TM7 originated from sternum, liver, and spleen tumors. Sequences with the ADOL name were obtained from different ALV-J viral strains propagated in C/E CEFs. The 39 LTR of ALVs included the U3 and R regions corresponding to

RT nested PCR exogenous avian leukosis virus

47

Fig. 2. Nested PCR of exogenous ALV. Lane 1, 1 kb plus DNA ladder (Invitrogen, Carlsbad, CA); lane 2, ALV-A (RPL-40); lane 3, ALV-B (RAV-2); lane 4, ALV-C (RAV-49); lane 5, ALV-D (RAV-50); lane 6, ALV-E (RAV-60); lane 7, ALV-J (ADOL-Hc1); lane 8, CEFs. Amplification products of approximately 210 bp were obtained for DNA template of subgroups A, C, and J. Amplification of DNA templates from subgroups D and B produced products of approximately 175 and 220 bp, respectively. and cells were frozen and thawed twice. One hundred-microliter aliquots of tissue culture material were collected from each well and transferred to 96well plates for ac-ELISA detection. Avian leukosis virus ac-ELISA assay readings were performed following the manufacturer’s recommendations (IDEXX Laboratories, Westbrook, ME) except we used a sample to positive control absorbance ratio (S/P) cutoff value of 0.1. The second plate was utilized for nested PCR and RT–nested PCR analysis. One hundred-microliter aliquots of cell-free tissue culture supernatant were collected individually from the five replicates of each viral dilution and utilized for RNA extraction following the procedure described above. The remaining infected cells were scraped, and 100-ml aliquots were collected individually from five replicates of each viral dilution for DNA extraction following the procedure described above. Detections of ADOL-Hc1 by acELISA, nested PCR, and RT–nested PCR were compared. Detection of ALV-J in cloacal swabs by acELISA and RT–nested PCR. Detections of ALV from cloacal swabs by ac-ELISA and RT–nested PCR were compared. Twenty-five 6-day-old specific-path-

ogen-free embryos were injected in the yolk sac with the ALV-J strain ADOL-7501 at a titer of 103 TCID50/ml. Twenty-three of the 25 embryos hatched successfully, and at 1 day of age, cloacal swabs were taken and resuspended individually in 1 ml of phosphate-buffered saline with 0.1% Tween 80. Cloacal swab suspensions were frozen and thawed twice, and 100-ml aliquots from each cloacal swab suspension were transferred to 96-well plates for ac-ELISA detection. At the same time, 100-ml aliquots were collected for RNA extraction and further RT–nested PCR analysis. ALV ac-ELISA assay readings were performed following the manufacturer’s recommendations (IDEXX Laboratories) with a positive S/P cutoff ratio value of 0.2. Detection of ALV from plasma samples by virus isolation, ac-ELISA, and RT–nested PCR. A total of 384 plasma samples from four different flocks were subjected to virus isolation on secondary CEFs obtained from C/E embryos (Kestrel Inc.). Virus isolation procedures were performed as previously described by Fadly and Witter (11). Briefly, secondary C/E CEF cells were suspended in F-10/M199 medium containing 2 mg/ml of diethylaminoethyl at a cell concentration of 2.5 3 105 cells/ml. Fibroblasts

←

nucleotides 7517 to 7771 of HPRS-103 sequence (accession number Z46390). Sequences were distributed in three main clades. One includes sequences from exogenous ALVs A, B, and J and RSV subgroups A, B, C, and D. The second clade includes sequences from endogenous RAV-0, and in separate branches are LTR remnants of endogenous sequences evj, E33, and E51.

48

M. Garcı´a et al.

were transferred to 24-well plates and incubated until a 50%–70% confluent monolayer was observed; subsequently, each well was inoculated with 50 ml of plasma sample, and the plate was gently rocked to promote viral absorption and then incubated. Medium was changed 24 hr after initial incubation, and cultures were maintained in the incubator for a period of 7 days. After incubation, 40 ml of 5% Tween 80 solution was added to each well, plates were frozen and thawed twice, and aliquots of 100 ml of tissue culture material were collected for ac-ELISA assay. ALV ac-ELISA assay readings were performed following the manufacturer’s recommendations (IDEXX Laboratories) except we used a positive S/P cutoff ratio value of 0.1. In addition to the virus isolation, RT–nested PCR was performed on all 384 plasma samples. Separate aliquots from each plasma sample were stored at 280 C and first thawed for RNA extractions, performed on 100-ml aliquots. Statistical analysis. Sensitivity, specificity, and simple kappa coefficient values per flock were calculated with the use of formulas in SAS 8.02 (SAS Institute Inc., Cary, NC).

RESULTS Sequence analysis and primers design. Published sequences of the LTR U3 region for ALV subgroups A, B, and J; for Rous sarcoma virus (RSV) subgroups A, C, and D; for Rous associated virus (RAV-0); and endogenous LTR remnants were aligned with sequences obtained in this study for ALV-J isolates and tumor samples. Phylogenetic analysis (Fig. 2) indicated that ALV-J LTR U3 sequences were closely related to sequences of other exogenous ALV and RSV and more distant from U3 LTR sequences of endogenous viruses (Fig. 1). Within the ALV-J branch of the phylogenetic tree, some tumor sample sequences (TM3, TM7) were closely related to the U.K. prototype isolate HPRS-103 U3 sequence, whereas other tumor samples (TM1, TM2) were more closely related to U.S. isolates (ADOL-5369, 5569, 7501). On the basis of the LTR U3 sequence analysis, two pairs of nested primers were designed; primer sequences and locations are reported in Table 1. Primer Leu3.2F was designed as a degenerated primer in order to amplify the sequence of ALVs A, B, C, D, and J. This primer expands from imperfect repeat 1 to imperfect repeat 2 of elongation factor II of the U3 region (2). The other three primers possess sequences 98%–100% similar to ALVs A, B, and J (data not shown).

LTR primers specificity. Specificities of the external and internal nested primers was first tested separately. When purified DNA from exogenous ALVs A, B, C, D, and J, DNA from different chicken lines carrying endogenous loci and DNA from endogenous subgroup E virus were tested by PCR with either external or internal LTR primers, amplification products were observed only for exogenous ALVs A, B, C, D, and J templates. No amplification products were observed for endogenous virus RAV60 or any of the chicken line DNA templates (Table 2). As with single PCR, nested amplifications were specific for exogenous ALVs A, B, C, and J (Table 2). Fig. 2 shows an example of amplifications obtained with nested PCR. Size differences were observed among amplification products of exogenous ALV subgroups. Amplification products of approximately 210 bp were obtained for DNA template of subgroups A, C, and J, whereas amplification of DNA templates of subgroups D and B produced products of approximately 175 and 220 bp, respectively. Restriction enzyme analysis. Digestion of nested PCR products for ALV subgroups A, B, C, and J with restriction enzymes DdeI and TaqI are shown in Fig. 3. Amplification products of ALV subgroups A, B, and C remained uncut after digestion with DdeI (Fig. 3, lanes 1–3), whereas ALV-J PCR product was digested and produced two fragments of approximately 120 and 90 bp (Fig. 3, lane 4). On the other hand, amplification products of ALV A, B, and C subgroups were digested with TaqI (Fig. 3, lanes 5, 6, 7), whereas ALV-J amplification product remained uncut after digestion with TaqI (Fig. 3, lane 8). As with subgroups A, B, and C, subgroup D amplification product remained uncut with DdeI enzyme and was cut with TaqI enzyme (data not shown). Therefore, restriction fragment length polymorphism (RFLP) was observed for nested PCR amplification products of exogenous ALVs. RFLP analysis was used to differentiate ALV-J from other exogenous viruses in plasma samples from naturally infected flocks. Detection of ALV-J Hc1 strain by acELISA, nested PCR, and RT–nested PCR. Detection limits of nested PCR and RT–nested PCR were compared with ac-ELISA by serial dilutions of ALV-J Hc1 strain in cell culture. Results for each assay are reported as the number of positive replicas within each viral dilu-

RT nested PCR exogenous avian leukosis virus

49

Fig. 3. Restriction enzyme analysis of ALV nested amplification products. Lane 1, D-15 ladder (Invitrogen, Carlsbad, CA); lanes 1–4 show digestions with enzyme DdeI. Lane 1, ALV-A Research Poultry Laboratory Strain 40 (RPL-40); lane 2, ALV-B (RAV-2); lane 3, RSV-C (RAV-49); lane 4, ALV-J (ADOL-Hc1). Digestion of ALV-J (ADOL-Hc1) amplification product produced two fragments of approximately 120 and 90 bp, whereas other subgroups remained undigested. Lanes 5–8 show digestions with enzyme TaqI. Lane 5, ALVA (RPL-40); lane 6, ALV-B (RAV-2); lane 7, RSV-C (RAV-49); lane 8, ALV-J (ADOL-Hc1). Digestion of ALV-A and RSV-C produced two fragments of approximately 135 and 65 bp. Partial digestion was observed for ALV-B (RAV-2), the undigested product of 220 bp and two fragments of approximately 150 and 70 bp were observed. Lanes 9–12 are undigested nested amplification products. Lane 9, ALV-A (RPL-40); lane 10, ALV-B (RAV-2); lane 11, RSV-C (RAV-49); lane 12, ALV-J (ADOL-Hc1).

tion (Table 3). The detection limit for acELISA was observed at viral dilution 1023 for five viral dilution replicates. The detection limit of Hc1 RNA by RT–nested PCR was observed at the 1024 viral dilution for one of five viral dilution replicates. The detection limit of Hc1 DNA by nested PCR was observed at the 1025 Table 3. Detection of ADOL-Hcl in C/E CEFs by ac-ELISA, nested PCR, and RT–nested PCR. ADOL-Hcl dilutions 1021 1022 1023 1024 1025 1026

ac-ELISA Nested PCR 5/5A 5/5 5/5 0/5 0/5 0/5

5/5 5/5 5/5 5/5 1/5 0/5

RT–nested PCR 5/5 5/5 5/5 1/5 0/5 0/5

Number of positive samples/replicas per virus dilution. A

viral dilution for one of five viral dilution replicas. Detection of ALV-J in cloacal swabs by ac-ELISA and RT–nested PCR. Detection of ALV-J by ac-ELISA and RT–nested PCR on cloacal samples is shown in Table 4. From a total of 23 samples obtained from ALV-J in ovo infected birds, 19 were positive by ac-ELISA as indicated by an S/P $0.200, whereas 13 were positive by RT–nested PCR. Eighty-three percent of the cloacal samples from ALV-J–infected birds were positive by ac-ELISA, whereas 56% were positive by RT–nested PCR. Detection of ALV-J in plasma samples by virus isolation (VI) and RT–nested PCR. VI and RT–nested PCR results on 384 plasma samples from three grandparents and one pedigree flock are shown in Table 5. Totals of 20, 19, 48, and 21 plasma samples from flocks A, B, C, and D were ALV positive by VI in C/E CEFs whereas 43, 45, 74, and 36 plasma sam-

50

M. Garcı´a et al.

Table 4. Detection of ALV-J by ac-ELISA and RT–nested PCR in cloacal swabs from in ovo infected birds.

S/P rangesA

Number of samples within S/P range

RT–nested PCR

0.000–0.1999 0.200B–0.999 1.000–1.599 1.600–1.999 2.000–2.999 % Positive

4 6 4 7 2 83%

0/4 3/6 2/4 6/7 2/2 56%

A S/P 5 ([sample absorbance] 2 [average negative control absorbance])/positive control absorbance. B S/P cutoff.

ples from flocks A, B, C, and D were identified as ALV-J positives by RT–nested PCR (Table 5). Restriction enzyme analysis was performed on all amplification products. Digestions with DdeI and TaqI produced RFLP patterns characteristic of ALV-J as shown above (Fig. 3). Because VI in C/E CEFs coupled with acELISA is considered the standard test for detection of exogenous ALVs (9), RT–nested PCR results on plasma samples were compared with VI (Table 5). True-positive (TP) plasma samples (VI1/PCR1) ranged from 13 to 43 samples among flocks. True-negative (TN) plasma samples (VI2/PCR2) ranged from 22 to 56 samples among flocks. False-negative (FN) plasma samples (VI1/PCR2) ranged from one to four samples among flocks. False-positive (FP) plasma samples (VI 2 /PCR 1 ) ranged from 17 to 30 samples among flocks. Notably, 30 VI2/PCR1 samples were observed for each of flocks A, B, and C (Table 5).

The sensitivity (SE) of RT–nested PCR to detect ALV-J RNA in plasma samples as compared with VI ranged from 79% to 94% among flocks, whereas the specificity (SP) ranged from 42% to 74% (Table 5). The level of agreement between VI and RT–nested PCR was expressed by the simple kappa (k) coefficient. A simple k coefficient of 1 indicates a perfect agreement between assays; a k coefficient of 0 indicates no agreement between assays. Agreement coefficients of k 5 0.3 and k 5 0.5 were obtained among the four flocks for VI and RT–nested PCR analysis of plasma samples. DISCUSSION

Initial sequence analysis of the 39 LTR region of ALVs indicated that this region was conserved among subgroups and among ALV-J strains and, therefore, was considered an appropriate target for amplification of ALVs. The most relevant characteristic concerning further application of the assay in the field was the specificity of the primers. As established in this study, LTR from ubiquitous endogenous virus subgroup E as well as DNA from chicken lines carrying endogenous virus loci were not amplified by either set of primers, and only different strains of ALV-J as well as strains of exogenous ALV subgroups A, B, C, and D were amplified. Amplification of exogenous viruses other than ALV-J was considered advantageous because this assay can also be utilized to detect and differentiate other exogenous viruses that have detrimental effects on poultry (18). To examine the nested reaction sensitivity, we compared the detection of ALV-J Hc1

Table 5. Detection of ALV-J in plasma samples by VI and RT–nested PCR. Positive samples Flock (age)

No. samples

A (37 wk) B (26 wk) C (16 wk) D (40 wk)

100 100 98 86

VI

RT– nested PCR

TP

TN

FN

FP

SE

SP

20 19 48 21

43 45 74 36

13 15 43 19

56 51 22 48

1 4 3 2

30 30 30 17

93 79 94 91

65 63 42 74

Comparison of assaysA k

0.3** 0.3** 0.5** 0.5**

TP 5 true positive, number of samples VI1/PCR1; TN 5 true negative, number of samples VI2/PCR2; FN 5 false negative, number of samples VI1/PCR2; FP 5 false positive, number of samples VI2/PCR1; SE 5 % sensitivity, TP/(TP 1 FN) 3 100; SP 5 % specificity (TN/(FP 1 TN) 3 100; k 5 kappa coefficients, measure of agreement between virus isolation and PCR (** P , 0.0001). A

RT nested PCR exogenous avian leukosis virus

DNA, RNA, and p27 antigen by nested PCR, RT–nested PCR, and ac-ELISA and found that both PCR procedures have detection limits superior to ac-ELISA. Although detection limit is not an accurate estimation of sensitivity, PCRbased methods apparently are more sensitive than ac-ELISA to detect ALV-J Hc1 strain in cell culture. Previous studies have found that the presence of ALV-J in cell culture can be detected earlier by PCR than by ac-ELISA (23). Several reports on the application of PCR for detection of ALV-J from whole blood (24), peripheral blood monocytes (23), and feather tips (24) found that PCR was more rapid, specific, and sensitive than other conventional diagnostic tests to detect ALV-J. In this study, with the use of RT–nested PCR, ALV-J was detected in 56% of the cloacal samples from in ovo infected birds, whereas 83% of the samples were positive by ac-ELISA. Given that cloacal samples were tested directly by ac-ELISA without prior VI, the exclusion of any present endogenous virus was not permitted. Therefore, our results suggest that RT–nested PCR may have excluded the detection of endogenous viruses, emphasizing the specificity of RT–nested PCR for the amplification of exogenous ALVs in clinical material as well. On the other hand, compared with VI, falsepositive reactions were observed with RT–nested PCR in plasma samples from naturally infected flocks (Table 5). These results raised questions regarding the RT–nested PCR SP in plasma samples. The possibility of PCR contamination was prevented by performing preand post-PCR procedures in separate rooms. In addition two to four negative controls were processed within each set of plasma samples to detect contamination throughout the process. Furthermore, the one tube RT–nested PCR provided an advantage over other conventional nested procedures by eliminating the need to transfer PCR product from the first amplification into a second amplification tube, a step that, if not handled properly, can become a major source of contamination during nested PCR procedures. To further test the specificity of RT–nested PCR in plasma samples, detection of ALV-J in plasma samples by RT–nested PCR and RTPCR assay with H5-H7 primers was compared (24). By these two PCR assays, ALV-J RNA was detected in 28 plasma samples by RT–nested

51

PCR, whereas ALV-J RNA was detected in 25 plasma samples with H5-H7 RT-PCR assay (data not shown). These results further corroborated that LTR nested primers are specific for the amplification of ALV-J RNA in plasma samples. Therefore, lack of specificity of the LTR primers was not considered the reason for the significant number of PCR false-positive (VI2/PCR1) plasma samples observed among 16-, 26-, 37-, and 40-wk-old flocks analyzed in this study (Table 5). Witter et al. (29) provided a detailed study on individual bird responses to ALV-J infection for VI, viral antigen, and antibody. In that study, the responses were categorized in four main ALV-J infection profiles identified as consistently positive, transiently positive, intermittently positive, and negative. In particular, VI responses of intermittently and transiently positive birds were characterized as inconsistent from weeks 12 to 62 after hatch. On the other hand, virus-neutralizing antibody responses were common among intermittently and transiently infected birds between 20 and 40 wk of age (29). Detection of ALV-J antibodies was performed on 17 plasma samples, identified as false positive, from flock D40 by ELISA (IDEXX Laboratories); ALV-J antibodies were detected in 14 of the 17 plasma samples (data not shown). Antibody detection was done on a limited number of plasma samples, and the capacity of the antibody response to neutralize virus was not examined. The presence of antibodies on 14 VI2/PCR1 plasma samples suggests that these birds presented either intermittent or transient ALV-J infection profiles. Although VI and RT–nested PCR assays were performed considering all technical aspects to assure the highest specificity and sensitivity of both assays, the level of agreement between both assays as applied to plasma samples ranged from low (k 5 0.3) to moderate (k 5 0.5). Therefore, rather than technical aspects, the lack of agreement between both assays most likely reflects the presence of transient and/or intermittent ALV-J infections characterized by highly inconsistent VI responses (29). The sensitivity of RT–nested PCR as compared with VI from plasma samples was also evaluated; 10 false-negative reactions were observed by RT–nested PCR. Some of the factors that might contribute to false-negative reactions

52

M. Garcı´a et al.

by RT–nested PCR may have been poor sample collection, poor nucleic acid extraction, or degradation of nucleic acids during storage. The one tube RT–nested PCR is a sensitive method for the detection of ALV-J and other exogenous ALVs. Because this assay is less laborious, considerably faster, and, in some instances, more sensitive than VI, utilization of this assay during ALV eradication programs may be an advantage. The control of ALV mainly depends on the early detection of positive birds. However, further studies are necessary to determine why VI was not possible in plasma samples where ALV-J was detected by RT–nested PCR. Findings along these lines would allow a better understanding of the advantages of RT–nested PCR and would permit a more efficient use of PCR during eradication programs. REFERENCES 1. Bacon, L. D. Detection of endogenous avian leukosis virus envelope in chicken plasma using R2 antiserum. Avian Pathol. 29:153–164. 2000. 2. Bai, J., L. N. Payne, and M. Skinner. HPRS103 (exogenous avian leukosis virus, subgroup J) has an env gene related to those of endogenous elements EAV-0 and E51 and a E element found previously only in sarcoma viruses. J. Virol. 69:779–784. 1995. 3. Boer, G. F. D., and A. D. M. E. Osterhaus. Application of monoclonal antibodies in the avian leukosis virus gs-antigen ELISA. Avian Pathol. 14: 39–55. 1985. 4. Calnek, B. W. Lymphoid leukosis virus: a survey of commercial breeding flocks for genetic resistance and incidence of embryo infection. Avian Dis. 12:104–111. 1968. 5. Chomzcynski, P., and N. Sacchi. Single-step method for RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159. 1987. 6. Crittenden, L. B. Exogenous and endogenous leukosis virus genes—a review. Avian Pathol. 10:101– 112. 1981. 7. Crittenden, L. B., S. McMahon, M. S. Halpern, and A. M. Fadly. Embryonic infection with the endogenous avian leukosis virus Rous-associated virus-0 alters responses to exogenous avian leukosis virus infection. J. Virol. 61:722–725. 1987. 8. Davidson, I., and R. Borenshtain. The feather tips of commercial chickens are a favorable source of DNA for the amplification of Marek’s disease virus and avian leukosis virus subgroup J. Avian Pathol. 31:237–240. 2002.

9. Fadly, A. M. Isolation and identification of avian leukosis viruses: a review. Avian Pathol. 29: 529–535. 2000. 10. Fadly, A. M., and E. J. Smith. Isolation and some characteristics of a subgroup J-like avian leukosis virus associated with myeloid leukosis in meattype chickens in the United States. Avian Dis. 43: 391–400. 1999. 11. Fadly, A. M., and R. L. Witter. Oncornaviruses: leukosis/sarcoma and reticuloendotheliosis. In: A laboratory manual for the isolation and identification of avian pathogens. J. R. Glisson, D. E. Swayne, D. J. Jackwood, J. E. Pearson, and W. M. Reed, eds. American Association of Avian Pathologists, Kennett Square, PA. pp. 185–196. 1998. 12. Garcia, M., S. Riblet, N. Ikuta, and V. R. Lunge. Molecular diagnosis and differentiation of avian leukosis virus subgroup J (ALV-J). In: Proc. 135th Annual Convention of the American Veterinary Medical Association, Baltimore, MD. p. 183. Jul. 25–29, 1998. 13. McKay, J. C. A poultry breeder’s approach to avian neoplasia. Avian Pathol. 27:S74–S77. 1998. 14. McKay, J. C., and A. G. Rosales. Control and eradication of ALV-J. In: Proc. International Symposium on ALV-J and Other Avian Retroviruses, World Veterinary Poultry Association, Rauischholzhausen, Germany. pp. 248–255. 2000. 15. Payne, L. N. Biology of avian retroviruses. In: The Retroviridae, 1st ed., vol. 1. J. A. Levy, ed. Plenum Press, New York. p. 489. 1992. 16. Payne, L. N. History of ALV-J. In: Proc. International Symposium on ALV-J and Other Avian Retroviruses, World Veterinary Poultry Association, Rauischholzhausen, Germany. pp. 3–12. 2000. 17. Payne, L. N., S. R. Brown, N. Bumstead, K. Howes, J. A. Frazier, and M. E. Thouless. A novel subgroup of exogenous avian leukosis virus in chickens. J. Gen. Virol. 72:801–807. 1991. 18. Payne, L. N., and A. M. Fadly. Leukosis/sarcoma group. In: Diseases of poultry, 10th ed. B. Calnek, J. Barnes, C. Beard, L. McDougald, and M. Saif, eds. Iowa State University Press, Ames, IA. pp. 414– 466. 1997. 19. Payne, L. N., A. M. Gillespie, and K. Howes. Induction of myeloid leukosis and other tumors with the HPRS-103 strain of ALV. Vet. Rec. 129:447– 448. 1991. 20. Payne, L. N., A. M. Gillespie, and K. Howes. Myeloid leukaemogenicity and transmission of the HPRS-103 strain of avian leukosis virus. Leukemia 6:1167–1176. 1992. 21. Silva, R. F., A. M. Fadly, and H. D. Hunt. Hypervariability in the envelope genes of subgroup J avian leukosis viruses obtained from different farms in the United States. Virology 272:106–111. 2000. 22. Smith, E. J. Endogenous avian leukemia viruses. In: Avian leukosis. G. F. DeBoer, ed. Martinus Nijhoff, Boston, MA. pp. 101–120. 1987.

RT nested PCR exogenous avian leukosis virus

23. Smith, E. J., S. M. Williams, and A. M. Fadly. Detection of avian leukosis virus subgroup J using the polymerase chain reaction. Avian Dis. 42:375– 380. 1998. 24. 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. Development and application of polymerase chain reaction (PCR) tests for the detection of subgroup J avian leukosis virus. Virus Res. 54:87–98. 1998. 25. Spencer, J. Progress towards eradication of lymphoid leukosis viruses—a review. Avian Pathol. 13:599–619. 1984. 26. Venugopal, K. Avian leukosis virus subgroup J: a rapidly evolving group of oncogenic retroviruses. Res. Vet. Sci. 67:113–119. 1999. 27. Venugopal, K., K. Howes, G. S. Barron, and L. N. Payne. Recombinant env-gp85 of HPRS-103 (subgroup J) avian leukosis virus: antigenic characteristics and usefulness as a diagnostic reagent. Avian Dis. 41:283–288. 1997.

53

28. Venugopal, K., L. M. Smith, K. Howes, and L. N. Payne. Antigenic variants of J subgroup avian leukosis virus: sequence analysis reveals multiple changes in the env gene. J. Gen. Virol. 79:757–766. 1998. 29. Witter, R. L., L. D. Bacon, H. D. Hunt, R. E. Silva, and A. M. Fadly. Avian leukosis virus subgroup J infection profiles in broiler breeder chickens: association with virus transmission to progeny. Avian Dis. 44:913–931. 2000.

ACKNOWLEDGMENTS We thank Dr. A. M. Fadly and Dr. B. Silva for kindly providing ADOL avian leukosis isolates, Dr. L. D. Bacon for kindly providing blood from different ADOL chicken lines, Mr. Pinghua Liu and Mr. Luis Miguel Morales for their technical assistance, and Dr. John Maurer for critical review of the manuscript. This work was supported by grant 550 from the U.S. Poultry and Egg Association, Tucker, GA.