Exogenous and Endogenous Leukosis

Transcription

1 Exogenous and Endogenous Leukosis Virus Genes Lyman B. Crittenden U. S. Department of Agriculture Agricultural Research Service Regional Poultry Research Laboratory East Lansing, MI i -

2 The viral and host genes associated with avian leukosis viruses (ALV) are among the best understood genes that chickens can acquire either by infection or inheritance. The basic framework of the molecular structure of these genes and their products is known. In contrast, the biological effect of the expression of these genes on chickens as they are maintained on commercial farms is poorly understood. I will outline the molecular biology of these genes and then explore our knowledge of their effect on the chicken. Retrovirus Replication: ALV genes that are acquired by exogenous infection are introduced into chicken somatic cells by infection with ALV particles called exogenous viruses. These viruses belong to a class of RNA viruses called retroviruses because of their unique method of replication. The left panel of Figure 1 outlines the replication of exogenous retrovlruses. The viral genomlc RNA is introduced into the cell, where a viral enzyme, reversetranscriptase, transcribes it into complementary DNA that is thought to be circularized before integration into the host chromosome. Host enzymes, then, transcribe this proviral DNA into RNA of different sizes. The full length RNA becomes the genome of infectious progeny virus and all the RNAs serve as messengers for translation into viral proteins. Exogenous proviral DNA is introduced at random normally into somatic cells and thus rarely enters the germline and becomes inherited like host genes. In contrast, the host can inherit ALV genes called endogenous viral genes in the germline. The right panel of Figure 1 shows that no exogenous infection is necessary. The inherited viral genes can be either partially or completely expressed after transcription - 2 -

3 to RNA and translation to viral proteins, and in some cases complete virions are formed. These genes are usually integrated at specific locl and are inherited as dominant genes. Transmission: Transmission of exogenous and endogenous ALV from chicken to chicken parallels the mechanism of replication of each virus type. The left panel of Figure 2 illustrates horizontal and congenital transmission of exogenous ALV. Transmission to progenyis congenital and entirely from the dam. Dams usually become congenital transmitters by congenital infection from their dam. Rarely does horizontal infection lead to congenital transmission. In contrast, as illustrated in the right panel of Figure 2, endogenous viral genes are inherited from both parents as are Mendelian genes. Viral Genes and Oncogenes: The avian retrovlruses can be divided into two general classes: those that are found to naturally infect chickens and induce neoplasms, such as lymphoid leukosls, after a long latent period; and those that have been developed by rapid laboratory passage and induce neoplasms such as sarcomas and leukemias in a very short latent period. Figure 3 diagrams the RNA genomes of some of these viruses. ALV contains only genes necessary for virus replication. These are genes for the groupspecific antigens (gag); reversetranscriptase (pol); envelope antigens that are responsible for induction of neutralizing antibody (en v); and a presumably non-coding region involved in replication (_). SR-Rous sarcoma virus (RSV) has all the genes necessary for viral replication and in addition a region specific for the induction of sarcomas (src). In contrast, the BH strain of RSV has sr cc substituted for env and cannot be packaged into infectious virus unless the cell is also infected with an ALV to provide en. v gene products. Similarly, -3-

4 the oncogenes myb, erb, myc, and fps have replaced vital viral genes in other laboratory strains that induce pathologically distinct neoplasms. DNA homologous to only viral genes found in ALV and only oncogenes found in the highly oncogenic viruses can be located in uninfected host DNA by nucleic acid hybridization. As shown in Figure 4, the viral genes are inherited as a unit in the same linear order found in the virus, and the different oncogenes are inherited independently. Viral genes are found only in chickens and some jungle fowl and are polymorphic. DNA homologous to each oncogene is found in all birds studied and can be found in many mammals; its structure and location is apparently similar in each species. The oncogenes are thought to be necessary for normal development, but are well regulated so they are expressed only at specific times by the host. If they are inserted into a somatic cell at a new location by a retrovirus or activated at the wrong time or place in development, they can lead to abnormal development and cancer. The rest of my discussion will center on genetic variation in endogenous viral (e vv) genes and the effect their presence or absence may have on the chicken. Phenotypic Expression o fe v_enes: The first evidence that ev genes existed was the presence of ALV antigen or complete ALVlike virus in cells or chickens known to be free of exogenous virus infection. When the env gene was expressed, the product always had the antigenicity of subgroup E, a subgroup unknown among exogenous ALVs. Table 1 gives the phenotypes that can be detected by standard biological methods and the viral genes needed to code for these products. -4-

5 Genetic Segregation of ev Phenotypes: Each of the positive phenotypes has been shown to be inherited as a single dominant gene, each apparently located at a different locus. Table 2 gives data representing classic genetic analyses of genes controlling two different phenotypes (gs+chf + and V-E+). The segregation of both phenotypes in the F 1 and first and second backcross generations after crossing two inbred lines agreed with the hypothesis that singledominant genes controlled each phenotype. The classic genetic approach suggested single gene control, but it could not be determined if the segregation observed involved variation in viral structural genes or if regulatory genes controlled the expression of ev structural genes that are relatively uniform among chickens. Molecular Analysis of ev Gene DNA: The structure of genomic DNA can now be analyzed at the molecular level if specific DNA or RNA probes for the gene can be prepared. ALV genomlc probes consisting of RNA or DNA free of host nucleic acids can be prepared from purified ALV particles. The "Southern blotting" procedure can then be applied to host DNA extracted from chicken tissue free of exogenous ALV. Host DNA is cleaved by bacterial restriction enzymes that are known to cleave DNA strands at locations where specific nucleic acid sequences occur. Gel electrophoresis separates the resulting DNA fragments by size. The DNA is then transferred by blotting to a nitrocellulose sheet and hybridized with the radioactive ALV probe. The sheet is washed and exposed to X-ray film, and the developed film has "bands" where particular sized fragments of DNA homologous to the ALV probe are located. Several laboratories have shown that there was a great deal of -5

6 polymorphism in band pattern among chickens, but patterns were relatively uniform within inbred lines. Therefore, molecular analysis of the host DNA showed that there was variation in structure or location of e vdna. Dr. Susan Astrin fortunately found a specific restriction enzyme, Sacl, that cleaved inside the ev genes only once very near one end. Therefore, the blots had only one major band for each locus where the ALV DNA was located. Dr. Astrin then showed that these bands segregated llke single genes that she called ev loci. Segregation, then, represented the presence or absence of ev genes at a particular locus. Collaborative work among Dr. Susan Astrin, Dr. Harriet Robinson, Dr. Edward G. Buss, Dr. Eugene Smith and myself combined classic genetic mating analyses and virological assays for phenotype with molecular analysis of DNA to identify 12 locl and the phenotypes controlled by the presence or absence of ev genes at each (Table 3). In those cases where a phenotype could be detected by virological methods, individual embryos from test-cross matings were analyzed for both DNA and phenotype. The exact association between the presense of a specific DNA band and the positive phenotype suggested that the band represented the gene coding for the ev phenotype. In the case of e v2complete subgroup E virus was found in some embryos that had only the band representing ev 2,showing that this gene contained all the coding sequences necessary for complete subgroup E virus production. Restriction of ev Gene Expression: e vvgenes vary widely in their expression, from e vl that has no detectable expression to ev 2 that codes for complete subgroup E virus production. Even genes such as e vv2 are restricted in virus production compared to ALV genes -6-

7 introduced by exogenous infection. This restriction can be due either to structural alterations within the gene or to host regulation of expression. Detailed structural mapping of some e vgenes gave the results shown in Figure 5. Three e vgenes lacked detectable alterations, yet evl and ev9 fail to yield positive viral phenotypes. Therefore, evl and ev9 are either restricted by other host genes or structural alterations are undetectable. As expected, ev 2is complete, but is apparently restricted in level of virus production by the host. The other ev genes analyzed all had detectable deletions that could account for the lack of full expression observed (Figure 5). Origin and Evolution of ev Genes: e vgenes are found in chickens and some but not all jungle fowl, and are not found in other fowl. We have established a chicken line free of ev genes showing that ev genes are not a necessary part of the chicken genome. We think they have been introduced by germllne infection with exogenous ALV. Those genes that either have no effect or a positive effect on reproductive fitness have survived. Insertion of exogenous viral genes into the germllne should often lead to integration of all the sequence elements necessary for the efficient production of a complete virus homologous to the exogenous virus inserted, e vgenes, studied so far, Clearly do not code for efficient production of a typical exogenous virus as they exist today. Expression is restricted and env, when expressed, always belong to subgroup E, a subgroup not found among exogenous ALVs. Complete endogenous viruses also have a very low oncogenlc potential. Apparently restricted expression and low oncogenlclty have been favored in the evolution of the chicken. Effect of ev Genes on Economic Traits: Infection with exogenous ALV leads not only to a low incidence of lymphoid leukosls in adult - 7 -

8 birds but, more importantly, to a reduction in egg production and general livability in laying hens. The consequences of partial or complete e vgene expression on the chicken are much less obvious. Complete endogenous subgroup E virus infection leads to very low levels of neoplasms and may induce none. One experiment comparing endogenous and exogenous virus infection of the same kind of chicken is summarized in Table 4. This cross is known to produce very high levels of endogenous virus spontaneously. About 500 chickens were kept under isolated conditions for 730 days. Two died with tumors indistinguishable from lymphoid leukosis and three with other neoplasms. Inoculation of chickens from the same cross with RAV-I, an exogenous ALV, at one day of age resulted in nearly 90% mortality with neoplasms by 167 days of age. Therefore, subgroup E virus expression resulted in few neoplasms in a type of chicken highly susceptible to the induction of neoplams by exogenous ALV. I think these neoplasms were not caused by the endogenous virus. Collaboration with Dr. Jan Gavora and Dr. Robert Gowe showed that about 30.0% of the embryos from a group of Ottawa inbred and non-inbred stocks produced infectious subgroup E virus. Lines selected for productivity hada lower incidence than randombred control lines, suggesting that selection reduced the frequency of genes coding for complete endogenous virus production. We are currently studying the effect of specific e vgenes on productivity and immune response in chickens semi-congenic for evl, 2, and 3. In limited studies, effects of e vgenes have been undetectable. Further studies are needed to clarify the effects of e vgenes on productivity in conventional environments. It is clear that the expression of e_v genes does influence

9 response to exogenous ALV infection, a virus genetically related to ev genes. We have observed these effects in semi-congenlc and backcross matings where specific ev genes are segregating. I will illustrate these effects with results of experiments comparing the response to RAV-I, an exogenous ALV, in our ev gene negative chickens and our standard cross (1515X7 I) known to express several ev genes and produce endogenous virus. Two perhaps independent phenomena were observed. Table 5 shows the results of inoculating RAV-I at one day of age. By 70 days, 42% of the pure ev- chickens had died with a "wasting" disease typified by shrunken firm livers, acltes and atrophy of the bursa and thymus. None of these symptoms were found in 1515X71 chickens. A mild anemia and a reduction in growth rate was seen much more often in the inoculated ev - chickens than in the 1515X71 cross. The data we have collected to date indicate that infection of chickens that do not express ev genes are much more likely to develop these symptoms at a relatively early age than chickens expressing e vgenes after ALV infection. Secondly, chickens that lack e vgene expression usually develop neutralizing antibody and clear the blood of vlremla earlier than chickens expressing ev genes after day-old infection with exogenous ALV. Table 6 shows that virus tlters start down in the ev- chickens before 21 days of age, while they remain high in 1515X71 chickens. By 42 days, the ev- chickens had no detectable virus in their sera, while the 1515X71 chickens almost all did. Neutralizing antibodies also occurred earlier and rose to a higher titer in the ev-- chickens. These observations are consistent with the idea that chickens expressing ev antigens as embryos are immunologlcally tolerant to -9-

10 antigens that are known to be shared by exogenous and endogenous viruses. This "partial" tolerance may interfere with maximum antibody production by chickens expressing ev genes. These data suggest that in field situations, where infection with exogenous ALV is common, ev gene free chickens would exhibit more non-neoplastic pathology than conventional chickens, but that horizontal exposure would lead to an early intense immune response that would usually prevent contact exposure from leading to congenital transmission. Therefore, ev negative chickens may be relatively easy to maintain free of exogenous ALV infection. Conclusions: Infection of chickens or their ancestors with exogenous ALV's has occasionally led to spontaneous integration of ALV DNA proviruses into the chicken germline. These endogenous viral (ev) genes segregate as dominant genes, but are not necessary for survival. The ev genes presently found in the germline are quite different from the DNA proviruses introduced into somatic cells by exogenous ALV infection. This suggests that the surviving ev genes are present because they do not reduce the reproductive fitness of the chicken. Little direct evidence is available to suggest that these genes are detrimental or beneficial to ALV-free chickens that carry them. But, the expression of ev genes does have a strong influence on response to exogenous infection with the genetically related exogenous ALV. Chickens lacking ev gene expression mount an earlier and stronger immune response and exhibit non-neoplastic pathology more often than chickens that express ev genes, after infection with exogenous ALV. The intense immune response of ev gene free chickens may make them easier maintain free of exogenous ALV infection. Further work must be done before recommendations on altering ev frequency in commercial chickens can be made. - i0 -

11 References Crlttenden, L. B Exogenous and endogenous leukosls virus genes - A review. Avian Pathology 10: , Crittenden, L. B. and S. M. Astrin Genes, viruses and avian leukosls. Bio Science 31: Hayward, W. S. and B. J. Neel Retrovlral gene expression. Current Topics in Microbiology and Immunology 91: Robinson, H. L Inheritance and expression of chicken genes that are related to avian leukosis sarcoma virus genes. Current Topics in Microbiology and Immunology 83: ii-

12 RNA DNA l 1 CircularDNA O Integrated _ Integroted DNA RNA. DNA 1! (mrna) (Genomic RNA) (mrna) (Genomic RNA) / \ / \ RNA...,l"---- Protein Synthesis Proteins Virlon Assembly Protein Synthesis Proteins Virion Assembly Figure 1. Replication of exogenous (left panel) and endogenous (right panel) ret rovi ruses. Exogenous Virus Endogenous Virus Horizontal or Congenital Infectious Virus Transient Viremia Immunity to exogeneous Virus Lymphoid leukosis rare Genetic ord' Viral DNA, Infectious Virus // integrated gamete DNAin 0 _egg _sperm o Chronic Viremia Viremia or antigen expressed Immune tolerance to Immune tolerance to exogenous Virus endoqenous Virus Lymphoid leukosis common Lymphoid leukosis very rare Figure 2. Transmission of exogenous (left panel) and endogenous (right panel avian retroviruses

13 909 pal env c ALV,,,,,, qoq pal env- src SR-RSV,,,, go9 pol src c BH-RSV : : : ga9 pal myb AMV,,, AEV,,Agogerb (?) MC29 FSV,&(Jog myc :Pg...tP... / Figure 3. Genetic maps of the RNA genomes of several avian retroviruses. The single line represents regions necessary for viral replication and assembly. The cross-hatched regions represent regions specific for oncogenic transformation. f "5.40 x 106 dalfons I.. i I _ Imegrated LTR 9a9 pal env LTR Viral DNA onc Integrated OncogenicDNA Host DNA 1-11 Large Terminal Repeat _--Viral DNA x'_-,_,_host Oncogenic DNA Figure 4. Genetic maps of inherited DNA homologous to viral sequences, endogenous viral (ev) genes, and viral oncogenic sequences, oncogenes

14 9o9 poi env,_ ev go9 pol env QO9 pol QoQ pol env c pol env c go9 P :\_._ po, env c gog : Cellulor sequences r_]r_ = Lon9 lerminol repeot (LTR) Figure 5. Maps of DNA of 7 ev genes showing the locations of deletions where detected. Table I. Viral phenotypes recognized as the expression of ev genes. Endogenous viral genes Phenotype expressed Symbol Expression of, env gs-chf* : :: subgroup E viral envelope antigen Coordinate expression of viral group- gag. env gs. chf _ spedf antigen and subgroup E viral envelope antigetn Inducibility of a viral particle unique to gag. pol V-15B* line15e Spontaneous complete V-E " proclucoon of genome subgroup E virus No Oetectable. none gs-chfexpr_ :L

15 Table 2. Classic genetic analyses of two ev phenotypes gs+chf + and V-E +. % Positive Mating Group-specific antigen RAV-O (V-E+)t (lines 7z (gs+)" (lines I and C) and 15s) PI P Pl BCI (P=) 40.0 (50% expected) 55.0 (50% expected) BC= (P2) homozygous (gs- or V-E-) 0.0 (11)_: (0% expected) 3.4 (6)t (0% expected) BC2 (P2) heterozygous (gs. or V-E*) 48.3 (9) (50% expected) 53.6 (6) (50% expected) P+ = Reaseheath line I; P2 = Reaseheath linec (Payne and Chubb 1968). tp, = RPRL line 72; P2 = RPRL Line 15B (Crittendenet al. 1977). tnumber of BCz parents presumed to be homozygousrecessive. Number of BCz parents presumed to be heterozygous. Table 3. ev!oci identified that co-segregate for DNA restriction enzyme patterns and the indicated ev phenotype. Locus Associated phenotype ev 1 chf-gsev 2 V-E + ev3 gs+chf + ev 4 chf-gsev 5 chrgsev 6 gs-chf + ev 7 V.15e* ev 8 chrgsev 9 gs-chf + ev 10 V-E*" evll V.E +- ev 12 V.E +- "I:Achlocus appears to code for II slightlydifferent subgroup E virus

16 Table 4. Mortality with neoplasms in a cross (15BX72) that is known to continually produce high levels of endogenous virus with'and without inoculation with the exogenous ALV, RAV-I. Days at No. Z Lot!/Inoc Sex risk obs. LL _ ONe* 1 None _ None _ None _ + _ RAV.1 _ + _ , * LL = lymphoid leukosis *_ ON = other neoplasms 1--/ Inoculated at 1 day Table 5. Mortality to 70 days of age in chickens lacking e v genes (ev-xev-) and expressing ev genes (1515X71) after inoculation at 1 day with RAV'-T1. d m Hating Number Z X 9 Chicks NNS Survivors NNS ev-xev I 5 X * NNS = a typical non-neoplastic syndrome Table 6. Exogenous virus isolations and titers in the serum of chickens lacking ev genes (ev-xev-) and chickens expressing evgenes fr---om7 to 1_-2da'ys after inoculation at 1 day with RAV-1. Hating log10 ticers +/total # X _ 7d 14d 21d 7d 14d 21d 28d 35d 42d ev-x_v /19 20/ /16 1/20 0/17 15I 5 X /20 19/19 20/ /

17 "Exogenous and endogenous leukosis virus genes" Questions and Answers I. G. B. Havenstein Have you looked at the effects of exogenous LL virus on the livability and performance of a stock in relation to whether or not the stock had received adequate maternal antibody protection for IBD (assuming, of course, that the chicks were exposed to the infectious IBD virus)? L. B. Crittenden We have not looked at the effect of maternal antibody to IBDV or the effect of exogenous leuksis virus on livability and performance. 2. G. B. Havenstein What effect does damage to the immune system by the IBD virus have on susceptibility to horizontal exposure to LL and subsequent vertical transmission of LL to the next generation? L. B. Crittenden Dr. Aly Fadly has conducted experiments on the effect of IBDV infection in chicks on the proportion that shed virus into their swabs after contact infection with exogenous leukosis virus. Such infection does increase the frequency of shedding and presumably of transmission to the next generation. We think immunodepression by IBDV lowers the ability of the chicken to respond immunologically to the leukosis virus. Such infections may have practical implications in leukosis virus reduction programs. 3. A. W. Nordsko$ What is the situation today as to what is known regarding the frequencies of subgroup A, B, C and D in commercial flocks? L. B. Crittenden Subgroup A is the most frequently isolated virus from commercial chickens. Subgroup B is much less frequent and subgroups C and D very rare. 4. K. Goodwin Are all known endogenous viruses of type E? Do type E viruses occur in exogenous form? L. B. Crittenden All endogenous leukosis viruses isolated to date belong to subgroup E. Infectious virus originally coded for by endogenous viral genes can infect subgroup E susceptible cells and chickens in the same way as an exogenous virus

18 5. R. N. Shoffner There is some evidence of leukosis in turkeys. Are the same types of retrovirus involved as in chickens? Is there any evidence for endogenous virus production in this species? L. B. Crittenden As far as we know, the avian leukosis viruses do not spontaneously infect turkeys and induce tumors. Turkeys are susceptible to artificial infection with subgroup A, but limited data indicate that LL-like tumors are not induced. Two unrelated retroviruses are known to induce tumors in turkeys. These are reticuloendotheliosis virus that occurs in turkeys in the U.S. and other countries and lymphoproliferative disease virus that is unknown in the U.S. but accurs in Europe and Israel. There is no evidence for endogenous viral genes in turkeys related to any of these three viruses