MOLECULAR BIOLOGY Characterization of Four Endogenous Viral ...

MOLECULAR BIOLOGY Characterization of Four Endogenous Viral Genes in Semi-Congenic Lines of Meat Chickens1 A. A. GRUNDER,2 B. F. BENKEL,3 J. R. CHAMBERS,4 M. P. SABOUR,4 J. S. GAVORA, and J. W. DICKIE Centre for Food and Animal Research, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada, K1A 0C6 ABSTRACT Restriction fragment length polymorphism analyses were used to examine endogenous viral genes (ev genes or ALVE genes) of the avian leukosis viral (ALV) family in semi-congenic lines of meat chickens. The Generation 6 lines examined in this study were semi-congenic in that each contained birds with either zero or with one ALVE gene in hemizygous state plus some solitary long terminal repeat (LTR) elements. Using four restriction enzymes on chicken genomic

DNA and two probes, one representing the entire ALV retroviral genome and one with only a small part plus the LTR, four ALVE genes were characterized. Each seemed to be complete with no detectable deletions. None appeared to be similar to known ALVE genes of White Leghorns, whereas two of the four may be the same as ALVE genes reported by others in White Plymouth Rock chickens.

(Key words: meat chickens, endogenous viral genes, semi-congenic lines, characterization, polymorphism) 1999 Poultry Science 78:873–877

chickens. Such genes have also been found in commercial broiler strains (Boulliou et al., 1991). Because exhaustive comparisons between labs are difficult, the exact number of ALVE genes is unknown but could exceed 50. Because most birds carry several ALVE genes, as many as one to five loci in noninbred Leghorns (Rovigatti and Astrin, 1983; Aarts et al., 1991) and an average of 7.3 loci in meat birds (Sabour et al., 1992), study has been difficult using the Southern blot technique. To expedite investigation of ALVE genes in meat-type chickens, Grunder et al. (1995) developed semi-congenic lines, within which each chicken carried none or one ALVE gene in the hemizygous state. They reported eight ALVE genes, seven of which had not been observed in White Leghorn strains. The present study was directed at describing four more ALVE genes derived from meat-type birds by utilizing such semicongenic lines to discriminate between different ALVE loci.

INTRODUCTION Endogenous DNA viral elements are apparently ubiquitous in the animal kingdom. The elements are genes of retroviral origin and are transmitted in a mendelian fashion. At least three families of retroviral elements have been described in chickens, including ALVE, which comprises proviruses closely related to Avian Leukosis Viruses, the ART-CH elements (Gudkov et al., 1992), and endogenous avian retroviruses (EAV) (Dunwiddie et al., 1986). The ALVE elements may be complete with the viral sequences gag, pol, and env flanked by long terminal repeats (LTR), or they may exhibit deletions for portions of these sequences (Rovigatti and Astrin, 1983; Crittenden, 1991). Some of the more complete of these elements may be transcriptionally active and produce viral proteins. Astrin (1978) was the first to study polymorphisms of what is now designated as ALVE genes (Benkel, 1998) using White Leghorn chickens. In a review, Crittenden (1991) reported 22 ALVE genes found in White Leghorn

MATERIALS AND METHODS

Semi-Congenic Lines Received for publication July 27, 1998. Accepted for publication January 16, 1999. 1Contribution Number 2475 from the Centre for Food and Animal Research. 2To whom correspondence should be addressed: Grunder@ EM.AGR.CA 3Present address: Lethbridge Research Centre, P.O. Box 3000, 5403 1st Avenue, S., Lethbridge, AB, Canada, T1J 4B1. 4Present address: Southern Crop Protection and Food Research Centre, Food Research Program, 43 McGilvray Street, c/o University of Guelph, ON, Canada, N1G 2W1.

These lines were developed such that each one contained only one ALVE gene to facilitate detection and

Abbreviation Key: ALV = Avian Leukosis virus; ALVE = endogenous provirus loci of the ALV type; ev = endogenous viral gene; EAV = endogenous avian retroviruses; IN = internal; LTR = long terminal repeat; PCR = polymerase chain reaction; RAV = Rous-associated virus; RFLP = restriction fragment length polymorphism.

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characterization using Southern blot and polymerase chain reaction (PCR) analyses. Thus, a semi-congenic line would contain birds, each of which had one ALVE gene in the hemizygous state, or none at all. The line was not necessarily free of solitary LTR elements. These lines originated from a WG Leghorn strain male (Gavora et al., 1989) that was free of ALVE genes except for the solitary LTR ALVE16 (Grunder et al., 1995) and mated to females of eight meat-type strains. The development of these strains has been described in detail by Grunder et al. (1995). The present study is based on birds of Generation 6 hatched in 1995.

Southern Blot Analysis Each analysis was carried out according to standard protocols described by Maniatis et al. (1982) in order to achieve interpretable restriction fragment length polymorphism (RFLP) on autoradiograms. High molecular weight genomic DNA samples were digested with the following restriction enzymes, one enzyme per sample: BamHI, EcoRI, HindIII, and SacI. Two probes were routinely used for hybridization: 1) the 8-kbp SalI fragment of pRAV-2, which represents the complete genome of the Subgroup B ALV virus, Rous-associated virus (RAV)-2 (Ju et al., 1980); 2) the 520-bp PCR fragment that contains the bulk of the 3′ LTR plus the C region of the element ALVE1 (Gavora et al., 1995). Probe 2 would be expected to hybridize with both the 5′ and 3′ junction fragments, although better with the latter because more of the sequences are in common. These two probes and their relation to the ALV genome are diagrammed in Figure 1. Other details of Southern blot analyses from digestion of high molecular weight genomic DNA to the production of autoradiograms to reveal RFLP and determination of band sizes can be found in Grunder et al. (1995).

Endogenous Viral Gene Detection by PCR The DNA of pedigreed birds was routinely screened for the presence of ALVE genes by rapid extraction of DNA from blood (Higuchi, 1989), then using a multiplex PCR test targeting the 3′ env-LTR region of the endogenous viral elements (Benkel et al., 1994). This test distinguished between birds that were completely free of subgroup E sequences, birds that carried a solitary LTR, and birds that carried at least one element that includes the 3′ portion of the env gene.

RESULTS AND DISCUSSION

Southern Blot Analysis An autoradiogram of a Southern blot of genomic DNA from four birds, each carrying a single ALVE gene, is shown in Figure 1. The SacI junction fragments shown in

Lanes 1 to 4 are designated ALVEB9, ALVEB10, ALVEB11, and ALVEB12, respectively. The BamHI junction fragments of these very same DNA samples are shown in Lanes 5 to 8, respectively. Hybridization with pRAV-2 detected both the 5′ and 3′ junction fragments as well as the internal element-specific bands in EcoRI and HindIII digests, but the data were not shown. However, in BamHI and SacI digests, the overlap between the pRAV-2 probe and the portion of the proviral genome associated with the 5′ junction fragments was too small to allow for the reliable detection of these bands. Therefore, in order to visualize 5′ junction fragments in BamHI and SacI digests, blots were stripped and rehybridized with the LTRspecific probe (data not shown). Table 1 summarizes the sizes of the restriction fragments observed with the four restriction enzymes used in this study. All four elements displayed two internal fragments (1.3 and 1.7 kbp) in BamHI digests, and one internal fragment of the expected size for complete elements for each of the EcoRI and HindIII digests, in addition to the element-specific junction fragments. Thus, within the resolution of the RFLP analysis, these elements appear to be complete between the upstream BamHI and the downstream HindIII sites as diagrammed in Figure 1. As for the 5′ end of elements, the bulk of the results indicated that the 5′ LTR were also intact for all four loci; however, it was not possible to detect 5′ junction fragments for all elements in all digests. For example, the 5′ junction fragment for ALVEB9 was not visible in SacI blots despite the use of the LTR-specific probe. However, because the 5′ junction fragments for this element were detected with the same probe for the three other enzymes used, the absence of the 5′ junction band in the SacI digests is almost certainly an artifact. Similarly, 3′ junction fragments were not detected for all elements in all digests (see Table 1). There are at least two possible reasons for these types of artifacts. Very large fragments do not transfer efficiently from the gel to the nylon membrane and can co-migrate with residual, undigested genomic DNA. Alternatively, on autoradiograms resulting from hybridizations with the LTR-specific probe, junction fragments may have comigrated with solitary LTR elements known to be in the birds and thus escaped detection.

Uniqueness of the Elements A survey of the literature was used to compare the molecular sizes of published RFLP fragments of loci with fragments of the four loci of the present publication. Loci ALVEB9 and ALVEB10 did not appear to be similar to those published previously. Locus ALVEB11 may be the same as the ev-cw1-10 or ev-cw2-8 loci reported by Aarts et al. (1992), in which the SstI fragment was 11.0 kbp, whereas the isoschizomer SacI used in the present study gave a fragment of 11.5 kbp (Table 1) in addition to a 1.6-kbp fragment not reported by Aarts et al. (1992). Their

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FIGURE 1. Restriction fragment analysis of meat chicken-derived elements. The top part of the figure shows a consensus map for avian leukosis virus subgroup E loci, based on the structures of Rous-associated virus (RAV)-0 and endogenous virus (ALVE1), along with two probes used for restriction fragment length polymorphism (RFLP) analysis in this study. Solid lines above and below the consensus map indicate the probes used for hybridization. Probe 1 represents the entire genome of RAV-2. Probe 2 contains 520 bp including the 3′ end of the env gene, the entire C-region and most of the 3′ long terminal repeat (LTR) of ALVE1. Restriction enzyme sites shown are: B, BamHI; E, EcoRI; H, HindIII; L, BglII; S, SacI. The rest of the figure shows an autoradiograph of a Southern blot of genomic DNA, digested with SacI (Lanes 1 to 4) or BamHI (Lanes 5 to 8) and probed with Rous-associated virus genome RAV-2 at high stringency. Content of Lanes 1 to 4 is the same as 5 to 8 and represents ALVE genes B9, B10, B11, and B12, respectively. Molecular weights shown on the left of the figure are based on HindIII digest of phage l DNA.

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TABLE 1. Restriction analyses of four meat chicken-derived avian leukosis virus type endogenous viral (ev or ALVE) elements using four restriction enzymes, sites of which are shown in Figure 1. Presence of internal (IN) fragments of expected sizes is indicated (Y). Sizes of 5′ and 3′ junction fragments are given in kilobase pairs. Isolated instances in which junction fragments were expected but not detected are indicated with a question mark BamHI

EcoRI

Locus

5′

IN1 (1.3)

IN2 (1.7)

3′

5′

IN (4)

ALVEB9 ALVEB10 ALVEB11 ALVEB12

25 3.4 12.6 ?

Y Y Y Y

Y Y Y Y

7.7 5.0 6.8 21.2

25.0 11.5 5.9 10.0

Y Y Y Y

loci were found in a White Plymouth Rock strain, assumed to be the major progenitor breed of the dam strains of meat chickens that contributed to the semi-congenic lines of our study. Locus ALVEB12, which produces an RFLP 3′ fragment of 7.5 kbp, may be the same as locus ev-cw1-16 of Aarts et al. (1992), who observed a 7.6-kbp band with SstI digestion of White Plymouth Rock DNA; however, they did not report the 15.1-kbp fragment that we observed in SacI digests or the 21.2-kbp fragment that we observed with BamHI. None of the four loci of the present study seem to be the same as any of the 12 described in a strain of Rhode Island Red chickens by Gorbovitskaia et al. (1998). There are two major recommended methods to establish identity of ALVE loci. One is to obtain the nucleotide sequence of the proviruses and compare one with the other. Another method is to develop specific PCR tests (Benkel, 1998) for a provirus and then compare assumed identical loci using this test. True comparisons of loci among laboratories await application of one or both of these methods. This report adds four more ALVE loci to the eight already found in our semi-congenic lines derived from meat-type strains. Of these 12 loci, only ALVEB7, equivalent to ALVE3, is definitely found in White Leghorn strains (Grunder et al., 1995). The description of these loci should facilitate further studies in explaining the reasons for continued segregations, including possible associations with production traits and disease resistance.

ACKNOWLEDGMENT The authors are indebted to the Greenbelt Research Farm poultry staff of the Centre for Food and Animal Research for performing the technical duties of reproducing and maintaining the chicken stocks.

REFERENCES Aarts, H.J.M., M. C. van der Hulst-Van Arkel, G. Bouving, and F. R. Leenstra, 1991.Variations in endogenous viral gene patterns in White Leghorn, Medium Heavy, White Plymouth Rock, and Cornish chickens. Poultry Sci. 70: 1281–1286. Aarts, H.J.M., M. C. van der Hulst-Van Arkel, and F. R. Leenstra, 1992. Different endogenous viral loci in Cornish and White Plymouth Rock chickens. Theor. Appl. Genet. 85:325–330.

HindIII 3′ 1.1 2.5 ? 23.0

SacI

5′

IN (3.5)

3′

5′

3′

29.3 7.2 4.0 4.1

Y Y Y Y

6.3 6.2 3.1 2.9

?

29.9 20.4 11.5 7.5

4.9 1.6 16.5

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ENDOGENOUS VIRAL GENES OF MEAT CHICKENS Ju, G. F., L. Boone, and A. M. Skalka, 1980. Isolation and characterization of recombinant clones of avian retroviruses: size heterogeneity and instability of the direct repeat. J. Virol. 33:1026–1033. Maniatis, T., E. F. Fritsch, and J. Sambrook, 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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Rovigatti, U. G., and S. M. Astrin, 1983. Avian endogenous viral genes. Curr. Top. Microbiol. Immunol. 103: 1–21. Sabour, M. P., J. R. Chambers, A. A. Grunder, U. Kuhnlein, and J. S. Gavora, 1992. Endogenous viral gene distribution in populations of meat-type chickens. Poultry Sci. 71: 1259–1270.