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Detection of reticuloendotheliosisvirus infection using the polymerasechain reactionMona M. Aly a , Eugene J. Smith a & Aly M. Fadly aa US Department of Agriculture , Agricultural Research Service,Avian Disease and Oncology Laboratory , 3606 East Mount HopeRoad, East Lansing, Michigan, 48823, USAPublished online: 12 Nov 2007.

To cite this article: Mona M. Aly , Eugene J. Smith & Aly M. Fadly (1993) Detection ofreticuloendotheliosis virus infection using the polymerase chain reaction, Avian Pathology, 22:3,543-554, DOI: 10.1080/03079459308418942

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Avian Pathology (1993), 22, 543-554

Detection of reticuloendotheliosis virus infectionusing the polymerase chain reaction

MONA M. ALY1, EUGENE J. SMITH2 & ALY M. FADLY

US Department of Agriculture, Agricultural Research Service, Avian Disease andOncology Laboratory, 3606 East Mount Hope Road, East Lansing, Michigan

48823, USA

SUMMARY

The polymerase chain reaction (PCR) was shown to be a sensitive and usefulmethod for detection of infection by members of the group of avian reticuloendothe-liosis virus (REV). Genomic DNA extracted from chick embryo fibroblasts (CEFs),blood and tumours of chickens experimentally infected with the spleen necrosis virus(SNV) strain of REV was used as the target for chain elongation. Deoxyoligonucle-otide primers that encompass a portion of the unique 3', repeat and unique 5' regionof the long terminal repeat (LTR) served to prime chain elongation. Productscharacteristic of the SNV LTR were also produced from DNA extracted from CEFinfected with other strains of REV, namely chick syncytial virus, duck infectiousanaemia virus, and the T-strain of REV. SNV-LTR sequences were amplified fromDNA of SNV-experimentally infected chicks between 5 days and 19 weeks afterinfection. SNV-LTR sequences were also amplified from DNA from tumours andbrains of SNV infected chickens, but not from DNA from avian leukosis virus- orMarek's disease virus-induced tumours. Results indicate that PCR is a sensitive andspecific method for detection of REV infection and tumours.

INTRODUCTION

Reticuloendotheliosis viruses (REV) comprise a group of avian type-C retro-viruses that induce reticular and lymphoid tumours in chickens, turkeys and quail(Witter, 1991). Members of this group include: defective, transforming virus(REV-T), replication competent helper, REV-A, spleen necrosis virus (SNV);chick syncytial virus (CSV), and duck infectious anaemia virus (DIAV). Depend-ing on the strain of REV and the line of chicken, REV can induce bursallymphomas that are similar to those induced by avian leukosis viruses (ALYs) ornon-bursal lymphomas that resemble those induced by Marek's disease virus(MDV) (Witter & Crittenden, 1979 and Witter et al, 1986). Observationsindicating nerve lesions associated with Marek's disease and bursal lymphomasassociated with avian leukosis can also be seen in chickens infected with REV

Received 9 October 1992; Accepted 14 December 19921This work was conducted while the senior author was on study leave from the Animal HealthResearch Institute, Cairo, Egypt.2To whom reprint requests should be addressed.

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confounding diagnosis. REV has also been implicated in necrotic dermatitis inchickens (Howell et al, 1982). Periodically, REV-infected flocks have beenreported and contaminated Marek's disease vaccines were associated with out-breaks of reticuloendotheliosis in Australia (Jackson et al, 1977) and Japan(Kawamura et al, 1976). Thus, an improved diagnostic assay for REV would alsofacilitate flock surveillance programs and certification of vaccines.

A complement-fixation test for REV (COFAR) has been described (Smith etal, 1977) but currently a REV indirect fluorescent antibody assay (Witter et al,1970) and an envelope-specific enzyme-labeled immunosorbent assay (ELISA)(Cui et al, 1988) are used to detect REV infection. Although sensitive, the latterprocedures require prolonged cultivation of virus on CEFs.

The polymerase chain reaction (PCR) presents a convenient in vitro method forthe specific amplification of targeted DNA. Since its introduction in 1985 (Saikiet al, 1985), the PCR technique has become a popular approach in clinicaldiagnosis (Ehrlich et al, 1991) and genome analysis (Rose, 1991). Because thenucleotide sequence of the large terminal repeat (LTR) of REV is known(Shimotohno et al, 1980), primers were selected for the elongation of targetproviral LTR sequences in DNA extracted from CEFs or from blood andtumours of chickens that were experimentally infected with various strains ofREV.

MATERIALS AND METHODS

Chickens

Chickens were Fi progeny of a cross between ADOL lines 15I5 and 7U Thebreeder flock was free from many avian pathogens including REV, ALV andMDV as determined by routine serological surveys.

Viruses

SNV, (Trager, 1959) an ALV-free, non-defective member of the REV group, waspropagated and titrated on ADOL line 0 CEFs and used to infect chicks. Todetermine the intra-group REV specificity of the PCR, other strains namely,REV-T (Robinson & Twiehaus, 1974), REV-MN (Paul et al, 1977), DIAV(Ludford et al, 1972), SNV (Trager, 1959), and CSV (Cook, 1969) werepropagated on line 0 CEFs for 7 days and host cell DNA was extracted by theprocedure described below.

Experimental design

In experiment 1, 40 day-old chicks were infected with SNV at a dose of 104

infectious units/chick. Ten additional chicks were maintained as uninoculatedcontrols in separate isolators. Blood was drawn from 10 chicks at 1, 2, 3, 4, and10 weeks post-infection (PI) and samples were tested for infectious virus in an

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DETECTION OF REV BY PCR 545

envelope glycoprotein-based ELISA REV (Cui et al., 1988). Tumours werecollected from dead chickens or from chickens killed at 19 weeks of age.

In experiment 2, 35 chicks were inoculated with 104 units of SNV at hatch.Blood was tested for the presence of REV at 1, 2, 3, 4, 5, 6 and 7 days PI.

Virus isolation

100 ul of serial 10-fold dilutions of blood ranging from 10 ~* to 10"5 was placedon line 0 CEFs in 35 mm dishes. Five replicates of each dilution were incubatedfor 7 days. 100 ul of detergent-lysed and freeze-thawed CEFs was placed inmicrotitre wells that were coated with a mixture of monoclonal antibodies(MC11A25 and MC11C237, each diluted 1:1000) specific for REV envelopeglycoprotein gp62 (Cui et al., 1988). The SNW antigen was detected using rabbitanti-REV serum and peroxidase conjugated anti-rabbit IgG (Cui et ah, 1988).

Indirect immunofluorescent detection of antibodies against SNV

CEFs grown in microtitre wells were infected with 200 fluorescent focus formingunits of SNV in each well. After 5 days of cultivation, acetone-alcohol-fixed CEFswere treated with 50 ul of 1:20 dilutions of chicken anti-REV sera. After thoroughwashing, the wells were treated with 50 ul of 1:20 dilutions of fluorescein-conju-gated anti-chicken IgG (Kirkegaard & Perry, Gaithersburg, MD) (Chen et al.,1987). CEFs were examined with a fluorescent microscope under Ploem illumi-nation at 100 x magnification.

Preparation of DNA

40 u/of blood was added to 1.0 ml of 0.01 M Tris-HCl (pH 8.0) extraction buffer(0.1 M NaCl and 0.001 M EDTA), to which 25 units of Pronase and 40 ul of10% sodium dodecyl sulfate (SDS) had been added. After digestion at 37°C for2 to 4 h, DNA was extracted with a 1:1 mixture of Tris-HCl buffer-saturatedphenol-chloroform. The aqueous phase was removed and re-extracted withphenol-chloroform. DNA in the supernatant was precipitated with two volumesof absolute ethanol and the pellet dissolved in 0.5 ml of sterile distilled water(Maniatis et al., 1982). For specificity studies, DNA was prepared from tumoursknown to be experimentally transformed with ALV or MDV. About 1 g of frozentumour was minced and DNA prepared as described above. In a similar manner,5 X 105 fluorescent focus forming units of SNV were placed in a 150 mm culturedish that was seeded with 106 primary CEFs. After 7 days cells were lysed andDNA was purified and diluted for PCR amplification. The concentration of DNAwas determined by measuring absorbance at 260 nm and template concentrationswere adjusted to 50 ug per ml.

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Oligonucleotide primers

Based on sequences of the proviral SNV-LTR (Shimotohno et al, 1980) severalprimer pairs were synthesized at the Macromolecular Structure Facility at Mich-igan State University. Aliquots from main stock solutions were diluted to 20 uMwith sterile distilled water. Sense primer 11 5'-CATACTGGAGCCAATGGTG-TAAAGGGCAGA-3;PR extends from nucleotide 282 to 312 and antisenseprimer 19 5f-AATGTTGTAGCGAAGTACT-3' extends from nucleotide 555 to573 of the long terminal repeat of proviral SNV. This set amplifies a product of291 base pairs (bp) spanning the unique 5' (U5), repeat (R) regions and 118 bpof the unique 3' (UR) region of the LTR.

Polymerase chain reaction

Amplification reactions comprised the following components in a total volume of50 pi: 10 mM Tris-HCl (pH 8.3), 50 mM KC1; 1.5 mM MgCl2; dATP, dCTP,dGTP and dTTP each at 200 uM; 1.5 units of Taq DNA polymerase(Boehringer Mannheim, Indianapolis, IN); primers (1.2 uM each) and 250 ng oftemplate DNA. Cycling parameters used were 94°C for 2 min for initial denatu-ration followed by 30 cycles at 94°C for 1 min, 55°C for 2 min and 72°C for 1min. A final elongation proceeded at 72°C for 6 min. Mixtures lacking templatewere routinely included as negative controls. Reactions were conducted in a MJRModel PTC-100 thermocycler (MJ Research, Watertown, MA).

Electrophoresis of PCR products

A 20 JJ.1 sample of each reaction mixture was added to 2 ul of gel loading buffer(0.25% bromphenol blue tracking dye in 25% Ficol). Bands were resolved in1.5% agarose after electrophoresis in 1 x Tris-Borate-EDTA buffer (TBE) for2-3 h at 100 volts. Products were stained with ethidium bromide (0.5 \lg per mlof gel). Molecular size markers represented multiples of 123 bp.

Hybridizations with labelled pSNV-LTR

PCR products were transferred to MagnaGraph nylon transfer membrane (Mi-cron Separations Inc., Westborough, MA) using 6 x SSPE (0.90 M NaCl, 60mM monobasic sodium phosphate and 6 mM EDTA, pH 7.4). Membranes werehybridized with 32P-labelled recombinant plasmid SNV-LTR using a randomprimer labelling method (Feinberg & Vogelstein, 1983).

Limit of detection of SNV

To determine the least amount of proviral SNV required for amplification,plasmid p60BSal which represents 7.7 kb of proviral SNV ligated into pBR322(O'Rear et al, 1980) was serially diluted and, after electrophoresis, products were

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DETECTION OF REV BY PCR

2 3 4 5 6 7 8 M

547

- 1 2 3

Figure 1. Amplification ofproviral SNV-LTR sequences using template DNA from REV-infectedline 0 CEF. Lane 1, no template; lane 2, uninfectedline 0 CEF; lane 3, REV-T; lane 4, REV-MN;lane 5, duck infectious anaemia virus; lane 6, spleen necrosis virus; lane 7, chick syncytial virus-in-fected CEFs; lane 8, DNA from blood of an SNV-infected chicken; and lane 9, molecular sizemarker.

transferred to a nylon membrane. Amplified LTR was detected by hybridizationwith a 32P-labelled plasmid that contained only the SNV-LTR.

RESULTS

REV group specificity of the PCR

In the presence of 200 uM concentrations of each of the four deoxynucleotides,the optimum concentration of magnesium ions and template was 1.5 mM and250 ng, respectively (data not shown). DNA extracted from CEFs separatelyinfected with five strains of REV yielded products with the expected size of 291bp whereas DNA from uninfected CEFs was unreactive (Figure 1). The productsalso hybridized with the radiolabeled SNV-LTR probe (data not shown).

Sensitivity of the PCR

To compare the sensitivity of the PCR with an ELISA-based SNV cultivationassay, blood was drawn from experimentally infected chicks 1, 2, 3, and 4 weeksafter infection with SNV. The PCR revealed amplification of the SNV LTR andthe SNV cultivation assay revealed high titres of SNV in all samples tested (Table1). In subsequent PCRs, when chickens were 10 weeks of age, sufficient SNVprovirus was present among three low-titre chickens (less than 100 infectiousSNV particles per ml of serum) for amplification of SNV-LTR sequences.Moreover, PCR amplification was detected among six chickens that did notseroconvert (Table 2 and Figure 2). At the termination of the SNV exposure trial,

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548 M. M. ALY ETAL.

Table 1. Comparisons between SNVamplification and the polymerase chain

reaction in detection of SNV infection

Weekpostinfection

Virusisolation3 PCRb

10/10 (ND)C

10/10 (5.5 ± 0.2)10/10 (6.0)10/10(5.1+0.3)

10/1010/1010/1010/10

'Virus isolation from 0.1 ml of blood; num-ber positive/number tested (mean logio oftitre. Endpoints determined in supernatantfluids using an REV gp62 EUSA

bNumber amplified/number tested using250 ng of target DNA.

TSI.D. = not done

when surviving chickens were 19 weeks of age, DNA from all of three chickenstested had sufficient target SNV provirus in their red cells to amplify the LTR(data not shown).

Because SNV proviral LTR was detected 1 week after exposure, an experimentwas conducted to determine whether SNV can be detected within 1 week afterinfection. At 5 days PI, amplification of the LTR was produced from DNA fromfour of five chicks tested and all five chicks at 6 days PI but in no chicks on days1 to 4 PI (Table 3).

Table 2. Detection of viraemia, SNV-LTR andantibodies in 10-week-old chickens infected with

SNV at hatch

Chicken

123456789

10

Total

Viros"titre

>6.51.61.64.55.66.56.52.81.65.0

PCRb

detection

++++++++++

10/10

Antibody'detection

_-++---++-

4/10

"Log10 titre of SNV in 1 ml of blood.b + = product seen on ethidium bromide-stained

gels."Determined in an indirect immunofluorescent

assay.

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DETECTION OF REV BY PCR 5 4 9

A 1 2 3 4 5 6 7 8 9 10 11 12 13 14

- 1230

- 123

Figure 2. (A)Detection of proviral SNV-LTR sequences in DNA from blood 10 weeks afterinfection. Lanes 1 and 2; template DNA from uninfected hatchmates; lanes 3-12, DNA fromSNV-infected chickens (1-10 in Table 2 respectively); lane 13, no template; and lane 14, molecularsize marker. (B) Hybridization with 32P-labelled recombinant plasmid pSNV-LTR after transferto a nylon membrane.

To determine the minimal number of copies of SNV detectable, serial 10-folddilutions of the p60B Sail clone of SNV were prepared for amplification of theLTR. Plasmid proviral concentrations ranged from 300 nanograms (ng) per jxl to30 attograms (10"18g) per ul in the presence of 250 ng of DNA derived from aSNV-free line 0 chicken. The visual end-point for stained bands corresponded to15 femtograms (fg) or 600 copies of SNV (Figure 3, lane 10) and southern blotsdetected products with only 1.5 fg of target SNV (data not shown).

Table 3. Comparative sensitivities be-tween SNV cultivation assay andpolymerase chain reaction in detection ofSNV within 7 days after infection with

SNV at hatch

Daypost

infection

1234567

Virus'titre

1.01.01.8 ±1.43.810.44.8 ±0.24.8 ±0.25.610.2

PCRb

detection

0/50/50/50/54/55/55/5

"Mean log,0 titres + SEM of SNV inblood from five chicks inoculated withSNV at hatch.

bNumber amplified/number tested.

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550

1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 3. Limits of detection of SNV-LTR sequences in DNAfrompSNV. Serial 10-fold dilutionsofpSNVDNA were used as the source of template. (A) In a reaction mixture of 50 \il the amountsof target SNVDNA were: lane 1, 15 \lg; lane 2, 1.5 \ig; lane 3, 150 ng; lane 4, 15 ng; lane 5,1.5 ng; lane 6, 150 pg; lane 7, 15 pg; lane 8, 1.5 pg; lane 9, 150 fg; lane 10, 15 fg and lane 11,1-Sfg.

A 1

— 123

Figure 4. Specific amplification of SNV-LTR sequences in DNA from tumours and brains ofSNV-infected chickens. (A) Lanes represent products amplified from 250 ng of the followingtemplates: lane 1, no template; lane 2, uninfected line 0 DNA; lane 3, avian leukosis virus-inducedbursal lymphoma; lane 4, Marek's disease virus-induced tumour; lane 5, SNV-infected brain DNA(chicken 5022); lane 6, SNV-infected bursal tumour (chicken 5022); lane 7, SNV-infected brain(chicken 5049); lane 8, bursal tumour (chicken 5049); lane 9, DNA extracted from blood (chicken5022); and lane 10, molecular size marker. (B) Hybridization with n p-labelled plasmid pSNV-LTR after transfer to a nylon membrane.

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DETECTION OF REV BY PCR 551

Detection of REV infection in tumours from experimentally infectedchickens

To determine whether SNV could be detected in other tissues, DNA wasextracted from lymphomas and non-tumourous brains. PCR revealed that the291 bp product was amplified from DNA from bursal lymphomas and non-tu-mourous brains (Figure 4, lanes 5-8) but not from DNA from lymphomas knownto be transformed by ALV or MDV. (Figure 4, lanes 3-4). The presence of ALVand MDV was confirmed using appropriate oligonucleotides, in PCRs (data notshown).

DISCUSSION

Unlike other RNA viruses, retroviruses replicate through RNA-dependent reversetranscription which leads to chromosomally integrated proviruses. The presenceof proviral copies of the retroviral genome thus facilitates direct detection fromhost cell DNA and obviates the preparation of complementary DNA. The resultsof polymerase chain reactions demonstrated herein illustrate advantages of in vitroamplification of targeted sequences in clinical diagnosis. Because of confoundinggross and microscopic pathology associated with other oncogenic avian viruses(Fadly, 1987), the PCR approach can provide compelling aetiologic evidence inthe diagnosis of ambiguous cases.

Cloned proviral SNV (p60B Sail) is 7.7 kbp in size or 5.1 x 106 daltons inmolecular weight (O'Rear et al., 1980). In serial 10-fold titrations that started at15 ug per 50 ul reaction mixture, the end-point for detection corresponded toabout 15 fg of SNV or 600 copies of pSNV. In the presence of 250 ng ofuninfected chicken genomic DNA, which is equivalent to 105 cells (2.4 pg DNAper diploid cell), one copy of proviral SNV can be detected among about 170otherwise uninfected cells. Moreover, hybridization of the southern blot with32P-labelled pSNV-LTR revealed that the sensitivity was enhanced at least 10-fold.

Apart from the specificity and sensitivity of PCR, individual samples can bereadily tested in mass-screening programs and because products are small, stainedproducts are seen after only a short period of electrophoresis without usinglabelled hybridization probes.

It is notable that detection of residual proviruses in peripheral blood cells alsoextended well beyond the period of seroconversion; products were detected 19weeks after inoculation. Residual proviral markers detected long after the initialinfection will thus inform breeders of the past history of REV exposure in parentalbreeding flocks.

Because the specificity of the PCR is determined by the nucleotide compositionof the primers, only DNA from REV infected tissues was amplified; DNA fromblood from uninfected chickens or from tumours of ALV- or MDV-infectedchickens was not amplified. Occasionally a spurious 1230 bp product was am-plified (Figure 4, lanes 2 and 3) but in subsequent reactions employing single

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primers, only primer 11 generated this product. Degenerate priming may reflectthe large (20°C) difference in melting temperature and size between primers.Subsequently, a 19-mer primer that lacked the last eleven nucleotides of primer11 and had a 20°C lower melting temperature was used with target DNAs thatpreviously yielded the 1230 bp product. Using the same annealing and am-plification protocol, the spurious product was not seen and only the 291 bpproduct was amplified with truncated primer 11 and primer 19 with DNA fromSNV-infected chickens (data not shown).

Apart from serologic and pathogenic differences noted among REV subtypes(Chen et al., 1987), sequences that distinguish REV subtypes remain unknown.Because the LTR is the main regulatory element in proviruses, perhaps differ-ences in nucleotide composition of REV LTRs may confer host cell tropisms. Theamplification of LTR sequences in DNA from the brain suggests that SNV mayinfect brain cells but contamination with adventitious cells infected with SNVcannot be ruled out. Peripheral nerve lesions were reported in chickens inoculatedwith REV-T (Witter et al., 1970). It would therefore be of interest to compareneurotropic activity of other REV strains or subtypes with LTR products usingthe PCR.

Additional applications of the PCR include: retrospective diagnosis using DNArecovered from formalin-fixed, paraffin-embedded tissue sections (Kallio et al.,1991); certification of avian vaccines, detection of REV proviral insertion into thegenomes of MDV (Isfort et al., 1992); and other avian viruses and qualificationof specific pathogen-free flocks. The possible residence of proviral REV sequencesin other avian species or parasites that infect chickens can also be addressed usingthe PCR.

ACKNOWLEDGEMENTS

We thank Cecyl Fischer for competent technical assistance, Linda Metro forpreparation of the typescript and Hsing-Jien Kung for providing pSNV-LTR.

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RESUME

Détection de l'infection par le virus de la réticuloendothéliose paramplification génique

Il a été démontré que l'amplification génique (PCR) était une méthode utile et sensible dedétection de l'infection par des membres du groupe du virus de la réticuloendothéliose aviaire(VRE). L'ADN génomique extrait de fibroblastes d'embryons de poussin, du sang ou detumeurs de poulets expérimentalement infectés avec la souche du virus de la nécrose de la rate(VNR) du VRE a été utilisée comme cible pour l'élongation de la chaîne. Les amorces dedeoxyoligonucleotide qui entourent une portion de l'unique 3', répété et unique région 5' dela répétition terminal long (LTR) ont servi à initier l'élongation de la chaîne. Des produitscaractéristiques du VNR LTR ont également été produits à partir de l'ADN extrait de

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fibroblaste d'embryon de poussin infecté par d'autres souches de VRE, à savoir le virassyncytial du poussin, le virus de l'anémie infectieuse du canard, la souche T du VRE. Desséquences de LTR de VNR ont été amplifiées à partir d'ADN de poussins expérimentalementinfectés par le VNR entre 5 et 19 jours après l'infection. Des séquences de LTR-VNR ontégalement été amplifiées à partir d'ADN de tumeurs et de cerveaux de poussins infectés parle VNR, mais pas à partir d'ADN de tumeurs induites par le virus de leucose aviaire ou celuide la maladie de Marek. Les résultats montrent que la P.C.R. est une méthode sensible etspécifique de détection de tumeurs et d'infection de VNR.

ZUSAMMENFASSUNG

Nachweis von Reticuloendotheliosevirus mit Hilfe der Polymerase-Kettenreaktion

Es wurde gezeigt, daß die Polymersase-Kettenreaktion (PCR) eine empfindliche und nützlicheMethode zum Nachweis von Infektionen durch Angehörige der Reticuloendotheliosevirus(REV)-Gruppe ist. Genom-DNS, die aus Hühnerembryofibroblasten (CEFs), Blut und Tu-moren von experimentell mit dem Milznekrosevirus (SNV)-Stamm des REV infiziertenHühnern wurde als das Ziel für die Kettenverlängerung verwendet. Desoxyoligonucleotid-Primer, die einen Teil der Unique 3'-, Repeat- und Unique 5'-Region des Long TerminalRepeat (LTR) umfassen, dienten zum Anregen der Kettenverlängerung. Für das SNV-LTRcharakteristische Produkte wurden auch aus DNS hergestellt, die aus CEFs extrahiert wordenwar, die mit anderen REV-Stämmen, nämlich Küken-Synzytiumvirus, Virus der infektiösenEntenanämie und dem Stamm REV-T infiziert waren. SNV-LTR-Sequenzen wurden ausDNS von experimentell SNV-infizierten Küken zwischen 5 Tagen und 19 Wochen nach derInfektion amplifiziert. SNV-LTR-Sequenzen wurden auch aus DNS aus Tumoren undGehirnen SNV-infizierter Hühner amplifiziert, aber nicht aus DNS aus Tumoren, die durchHühnerleukose-Virus oder Virus der Marekschen Krankheit verursacht waren. Die Ergebnissezeigen, daß die PCR eine empfindliche und spezifische Methode für den Nachweis vonREV-Infektionen und-Tumoren ist.

RESUMEN

Detección de la infección par el virus de la reticuloendoteliosisempleando la reacción en cadena de la polimerasa

En este estudio se demostró que la reacción en cadena de la polimerasa (PCR) era un métodoutil y sensible para la detección de una infección por miembros del grupo del virus de lareticuloendoteliosis aviar (REV). Se empleó como diana para la cadena de elongación el ADNgenómico extraido de fibroblastos de embrión de pollo (CEFs), neoplasias y sangre de gallinasinfectadas experimentalmente con la cepa virus de la necrosis esplénica (SNV) del REV. Los"primers" deoxioligonucleótidos que abarcan una porción de las regiones única 3', repetitivay única 5' del fragmeto repetitivo terminal largo (LTR) sirvieron para cebar la cadena deelongación. Se produjeron también productos caracteristicos del LTR del SNV a partir deADN extraido de CEF infectados con otras cepas de REV, en concreto virus sincitial del pollo,virus de la anemia infecciosa del pato y la cepa T del REV. Las secuencias SNV-LTR fueronamplificadas también a partir del ADN de pollos infectados experimentalments con SNV entrelos 5 dias y 19 semanas postinfección. Se amplificaron tambien secuencias SNV-LTR a partirde ADN de tumores y cerebros de gallinas infectadas con SNV pero no del ADN de neoplasiasinducidas por el viras de la enfermedad de Marek y el virus de la leucosis aviar. Los resultadosindican que el PCR es un métodoa sensible y especifico para detectar la infección por REV ylas neoplasias.

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