Induced liver tumour further support to a genetic marker with its high correlation with chorioallantoic membrane phenotypes in selected layer lines.

The conventional chorioallantoic membrane (CAM) phenotype assay was conducted using 11-day-old embryonatic eggs of white Leghorn strains, each inoculated with 0.2 ml of subgroup A Rous sarcoma virus (RSV) and subgroup C RSV separately containing 10(3)pfu ml(-1). Eggs were further incubated for hatching. Harvesting of CAMs for counting of pocks and monitoring chicks for liver tumour (LT) mortality during 4 weeks of post-hatching period were followed. The conversely associated phenotype (CAP) incidence i.e. CAM(+) LT(-) and CAM(-) LT(+) was observed in all three lines for both subgroup A and C virus infection. The LT deaths of chicks in all strains occurred within 21 days post-hatch irrespective of virus subgroups. The regression analysis of %LT death (transformed data) distributed within pock count range (PCR) basis was performed. The regression coefficients (b(i)'s) were found to be non-significant, indicating that %LT death did not correlate with number of particles that entered the cells because the chicks that had at least 25 pock counts in CAMs died with few exceptions. This study upheld the view that the CAM phenotypes (S and R) under the control of a pair of alleles, a(s) and a(r) at the tva locus and c(s) and c(r) at the tvc locus as reported extensively. Because of a high correlation coefficients between CAM and LT phenotypes [S and LT(+)] in respect of subgroup A (r = 0.72) and subgroup C (r = 0.81) infection, it is obvious that one could postulate a pleiotropic control of the two traits by the tva and tvc genes, respectively. Indeed fitting a 4-allele model in both tva and tvc locus, suggesting that CAPs are the indicator for nullifying the conventional 2-allele model proposed for the induced tumour expression phenotypes by leukosis sarcoma viruses.

Subgroup-A Rous sarcoma virus-induced growth stimulation of chick embryos infected via the chorioallantoic membrane.

Chicks that hatch from eggs containing group specific antigen (gs antigen) of lymphoid leukosis virus (LLV) subgroups, grow poorly. In our laboratory for more precise identification of LLV-of subgroup A (LLV-A) resistant and susceptible genotypes by progeny testing, the chorioallantoic membrane (CAM) assay in complemented by liver tumour (LT) assay, wherein Rous sarcoma virus (RSV) of subgroup A (homologous to LLV-A) was used. The present study was conducted in a light breed (White Leghorn) and also in a heavy breed (Rhode Island Red) to ascertain the effect of infection on embryonic growth by RSV subgroup A. Mean relative body weight (rbw) of infected LT negative chicks of either breed exceeded the control highly significantly (P < 0.01) by 2%. However, neither the dose of virus inoculated per embryo, nor egg size influenced the relative body weight of day old chicks (P > 0.05). No difference in relative body weight of LT positive and control chicks was observed.

The genetics of mortality and survival of broiler chicks infected as embryos by subgroup A Rous sarcoma virus.

A study using two high-performance broiler lines, a synthetic male line (SML) and a synthetic dam line (SDL), was undertaken to investigate the pattern of mortality due to induced liver tumours (LT) and the immune response to subgroup A virus inoculated via the chorioallantoic membrane (CAM) route. The distribution patterns of the four possible phenotypes were similar in both sire and dam lines. The occurrence of conversely associated phenotypes was about 30S, in both the lines. The percentages of CAM(+) LT(-) and CAM(-) LT(+) were 14.26%, and 14.46% in the dam line and 20.0% and 9.57% in the male line. The LT mortality was 30-50% in the birds with low pock counts, whereas it was 80-93% in the birds with high pock counts. The group specific antigen shedding status did not influence death due to LT. In birds in the high pock count group, 98% of deaths due to LT were completed by the sixth week, whereas in those in the low pock count group, death due to LT was spread over 24 weeks. The SDL birds survived better than SML birds in the high pock count groups. In both lines, about 20% of deaths occurred owing to non-specific causes. The average survival time after hatching before death from LT was 26 days, whereas that for non-specific death was 81 days.

Genetic analysis of Rous sarcoma virus (subgroup A) induced tumourigenesis and performance of synthetic broiler stocks in relation to group-specific antigen shedding.

An investigation was made using two high-performance meat lines, synthetic male line (SML) and synthetic dam line (SDL), to assess the susceptibility pattern in response to subgroup A Rous sarcoma virus (RSV) and the effect of group-specific (gs) antigen shedding in eggs, as evidence of avian leukosis virus infection effects on production and fitness traits. The ar gene frequency was observed to be at 0.68 +/- 0.04 and 0.66 +/- 0.06 for SDL and SML lines, respectively. Synthetic broiler lines were fivefold less sensitive to pock expression in chorioallantoic membranes (CAMs) as compared with a White Leghorn (WL-2A) reference line, in response to Bryan standard RSV (subgroup A). It was also observed that gs antigen shedding had neither influence on pock response in CAMs nor on induced liver tumour death. The prevalence of gs antigen shedding, however, fluctuated from generation to generation, ranging from 16.66 to 69.23% in SDL and 20.68 to 51.85% in SML, indicating an exogenous infection. Analysis of the relationship of gs antigen shedding in eggs with some fitness traits and overall flock production traits provided strong evidence that fitness traits such as fertility, hatchability, and production traits, such as 6-week body weight and egg weight, were significantly influenced by gs shedding.

The genetic control of susceptibility to subgroup D RNA tumour virus infection in commercial breeds of chickens.

The genetic control of resistance to RSV (RAV-50) in chick embryos inoculated via the chorioallantoic membrane route was studied in three breeds of chickens. The relative susceptibility to subgroup D infection was 0.01, 0.02 and 0.003 in broilers, White Leghorns and Bantam x Australorp crosses, respectively. The distribution pattern of putative dr gene frequencies were 0.80, 0.68 and 0.90, respectively, in the same three breeds. Liver tumours resulting from the virus infection were observed in a very few birds about 28 days after hatching.

Interrelationship between tumour virus receptors, A and C, coded by host cell genes in fowl.

Bryan Standard strain of Rous Sarcoma Virus (BS-RSV) of subgroup A and pseudotype of Bryan high titre RSV(RAV-49) of subgroup C and an equal mixture of subgroup A and subgroup C virus were inoculated to 11-day old embryos of white leghorn (WL), Australorp (AL) and f1 and f2 generations of crosses between WL and AL breeds of fowl to detect and estimate the interrelationship between tumour virus receptor coding host cell genes of tva and tvc loci. Linkage values estimated on a pooled sex basis were 0.08 +/- 0.03 and 0.10 +/- 0.03 for WL and AL breeds respectively and 0.09 on pooled breed basis. This clearly indicates that the tva and tvc loci are indeed closely linked in WL and AL breeds of fowl and supports the concept of using subgroup C virus to raise stocks resistant to subgroup A virus infection.

Expression of tumour phenotypes, hatchability and liver tumour mortality in two routes of inoculation in fowl.

A total of 350 and 200 eleven-day-olc embryos (pooled breeds) of twelve hatch replicates were inoculated with pseudotype of Bryan high titre, RSV(RAV-49) of subgroup C viz CAM (chorioallantoic membrane) and YS (yolk sac) route, respectively. An increase in hatchability (about 16%) and decrease in the incidence of CAM(+) [71%] and LT(+) [47%] phenotypes was noticed when inoculation was done via YS route as compared to the inoculation via CAM routes. A delay in LT(+) mortality was also recorded in YS route of infection. Chi-square analysis within a route basis indicated highly significant contingency (P < 0.01) in association of CAM infection phenotypes and LT incidence phenotypes for CAM route of infection in contrast to the YS route of infection.

Genetic control of cellular infection by subgroups A and C RNA tumour viruses in guinea fowl.

An investigation was carried out in guinea fowl to determine their susceptibility to infection by Rous sarcoma viruses of subgroups A and C. A standard dose of each subgroup virus was inoculated into 14-day-old embryos via the chorioallantoic membrane (CAM). On the 10th day after inoculation, 50% of the embryonic chorioallantoic membranes were harvested to assess their infection status (CAM(+) or (-)), while the rest were allowed to hatch. The hatchabilities of the embryos inoculated with subgroups A and C were about 50% and 57%, respectively. The relative sensitivities of guinea fowl to infection by viruses of subgroups A and C were observed to be 0.220 and 0.003, respectively, as compared to chickens (1.00). Mortality due to subgroup A virus-induced liver tumours (LT) was 54% and four phenotypic subclasses, namely CAM(+) LT(+), CAM(+) LT(-), CAM(-) LT(+) and CAM(-) LT(-), were observed in guinea fowl as in chickens. However, a higher incidence (31%) of conversely associated phenotypes, i.e. CAM(+) LT(-) and CAM(-) LT(+), were observed in guinea fowl. Mortality caused by subgroup A virus-induced liver tumours was first observed in inoculated guinea fowl keets during the 3rd week after hatching, and 93% of the mortality occurred within 6 weeks. The peak mortality occurred in the 4th week after hatching. The target organs for transformation were considered to be the liver and spleen because of the equal incidence of tumours in these organs. Males and females were equally likely to die from liver tumours.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic susceptibility of indigenous chicks to subgroup A Rous sarcoma virus inoculated via the chorioallantoic membrane.

An investigation was made using chicks of two Indian indigenous breeds of fowl, Kadaknath and Aseel, to ascertain genetic resistance to infection by Rous sarcoma virus of subgroup A. A standard inoculation dose of 0.2 ml virus containing 1000 pock forming units ml-1 was injected via the chorioallantoic membrane (CAM) into the 11-day-old embryos that were subsequently hatched. The sensitivity of the two indigenous breeds was compared with the highly susceptible exotic White Leghorn (WL) strain maintained in the laboratory. The Kadaknath breed was about three-fold and Assel, about six-fold less sensitive than the WL strain, indicating superiority of the indigenous breeds over the exotic breed of fowl. Most of the CAM-susceptible chicks died of liver tumour (LT) and most of the CAM-resistant chicks survived. However, conversely associated tumour phenotype subclass chicks, i.e. CAM-susceptible LT-negative chicks that survived and CAM-resistant LT-positive chicks that died, occurred consistently in the three breeds of fowl. Nevertheless, the overall survival potential of Kadaknath chicks measured up to 8 weeks post-hatching was greater than that of Aseel chicks. Neither transformation of embryonic tissue prior to hatching nor the visceral metastasis including liver conformed with the degree of CAM-infection as measured by number of pocks on CAMs.

Genetics of post-hatching survival potential of Australorp chicks infected as embryos by subgroup A Rous sarcoma virus: further support to 4-allele genetic model.

Embryos (II day-old) of Australorp breed were inoculated via chorioallantoic membrane (CAM) with subgroup A Rous sarcoma virus, and hatched subsequently. The post-hatch survival period in chicks was recorded upto the last chick that died by virus-induced liver tumour, which had a range from 3 to 50 days with an average of 13 +/- 8.7 days. The survival potential of progency tested Australorp parents selected on the basis of negative CAM-infection and those selected on uninoculated embryos, differed significantly (P less than 0.01) while maintaining an inverse relationship between liver tumour mortality and degrees of infection of CAMs. The homozygous susceptibles lacking either ar1 or ar2 or both alleles of the tva (tumour virus a) locus died within 7 days of post-hatching, supporting thereby 4-allele genetic model of tva locus recently proposed for the control of LT- and CAM-infection phenotypes.

Inheritance of fibrosarcomatous liver tumours in White Leghorn chicks inoculated vu the chorioallantoic membrane with subgroup A Rous sarcoma virus: A four-allele genetic model.

An investigation was made using White Leghorn fowl to study the genetic control of subgroup A Rous sarcoma virus-induced fibrosarcomatous liver tumours (LT) in chicks inoculated via the chorioallantoic membrane (CAM). A total of 723 CAM-inoculated embryos were hatched in three experiments. In Expt 1, CAM-susceptibility and LT-mortality were examined on a within sire family basis to ascertain the inter-relationship between the two parameters. In Expt 2, sires that had records of high or low LT(+) deaths in the families were selected to produce progeny within high and low incidence lines in the next generation to ascertain the amenability of the trait (LT death) to selection pressure. Survivors of LT-assay (Expt 1) were mated inter se in Expt 3 to study the inheritance of the two traits according to known or proposed genetic models. It was shown that LT mortality is a genetic trait because of its amenability to selection, with a high realised heritability (h(2)(R)= 1.16). In the three experiments, most CAM-susceptibles (S) died of LT(+), and most CAM-resistants(R) survived, but there were some conversely associated phenotypes i.e. S(LT-) and R(LT+). The conventional 2-allele model of the tva (tumour virus a) locus with pleiotropic effect, or a 2-locus model with linkage, were considered inadequate to explain the occurrence of conversely associated phenotypes on a within family basis. However, a 4-allele model of the tva locus showed a good fit to the results of this study.

Induced liver tumour deaths by subgroup A Rous sarcoma virus in chicks inoculated via chorioallantoic membrane, a genetic marker.

A study was made using two strains of light breed (White Leghorn strains, A and B) and four heavy beeds (Rhode Island Red, New Hampshire, Australorp, Columbian) to evaluate the breed difference in survival potential of chicks that were infected as 11-day-old embryos via chorioallantoic membranes (CAMs) with a subgroup A Rous sarcoma virus. Of the 1185 chicks hatched over multiple hatch-replicates, 845 chicks died rapidly of a fibrosarcomatous liver tumour (LT) with a peak mortality about 74% attained by the second week, post-hatch, in the heavy breeds and more than 90% by the second week in the light breed. The breeds did not differ in induced LT mortality when the chicks hatched from eggs that had at least 25 pock counts on CAMs, apparently genetically susceptible, i.e. 25 biologically active virus particles were enough to induce an unpreventable fatal LT. However, low pock-count on CAMs did not act as a pointer for predicting genetic resistance to infection because about 23% of chicks developed from eggs that had no pocks on CAMs, apparently genetically resistant, also died of LT, requiring further studies.

Cellular immunity in chicks expressing fibrosarcomatous liver tumor following embryo infection with subgroup A Rous sarcoma virus.

Bryan standard strain of Rous sarcoma virus (BS-RSV) of subgroup A was inoculated into heavy and light breeds of chicken embryos via chorioallantoic membrane (CAM) to ascertain cell-mediated immune response, as measured by a leukocyte migration inhibition (LMI) test. Chicks hatched from eggs with pock-positive CAMs were more likely to develop liver tumors than those hatched from eggs with pock-negative CAMs. Chicks that developed tumors usually had a positive cell-mediated immune response, and those that were negative for liver tumor were negative, based on the LMI test.

Hatchability and mortality following infection of chick embryos with subgroup a Rous sarcoma virus.

An investigation, using Rhode Island Red, New Hampshire, Columbian and Australorp, was conducted to study the correlation between chorioallantoic membrane (CAM)-infection phenotype to challenge with Bryan strain of Rous sarcoma virus (BS-RSV) of subgroup A and subsequent mortality, following hatching. A total of 806 CAMs and 438 chicks over nine hatch-replicates in three experiments provided evidence that infection via an inoculation of the CAM induced rapid and high mortality, about 72%, averaged over the four breeds, from a fibrosarcomatous liver tumour (LT) in CAM-susceptible chicks. In contrast, only 14% of chicks inoculated via the yolk sac died from liver tumours. Infection via the CAM reduced hatchability by about 28% (P<0.01) as compared to the hatchability of embryos infected via the yolk sac. About 21% of chicks which developed no pocks on the CAM after RSV inoculation developed liver tumours and died.

Genetic control of resistance to subgroup A and subgroup C tumour viruses in Rhode Island Red fowl: evidence for linkage between the tumour virus a (tva) and tumour virus c (tvc) loci.

A study, using the Rhode Island Red (RIR) strain of fowl maintained at Houghton Poultry Research Station, was made to investigate the genetic control of cellular response to infection with viruses of subgroups A and C. Family matings within the RIR strain and test-crosses between the RIR parents and White Leghorn (WL) parents of known ararcrcr genotype were set up to ascertain linkage between the tumour virus a (tva) and tumour virus c (tvc) loci. The results confirmed that in this RIR strain, the two loci, tva and tvc, control the cellular response to viruses of subgroups A and C, respectively, as reported in other breeds of fowl (WL and New Hampshire). As in WL fowl, the two loci are linked. The linkage value of 0-22 in the male sex agreed well with that reported in the WL male sex, indicating that the two loci are located in the same sites in homologous chromosomes in the two breeds. However, in the RIR strain, no sex difference in crossing over between the two linked loci was found, contrary to that reported in WL fowl where the absence of crossing over between the two loci was observed in the heterogametic female sex.

Further studies in genetic resistance of fowl to RSV(RAV O): evidence for interaction between independently segregating tumour virus b and tumour virus e genes.

The segregation of resistant and susceptible phenotypes in response to infection by RSV(RAV 2), RSV(RAV 50) and RSV(RAV 0), of avian RNA tumour virus subgroups B, D and E, respectively, was analysed in several test-crosses using chickens from the RPRL line 7-2, HPRS-synthetic line E and the Reaseheath line C. The results were fully consistent with out view reported previously that the genes at the tve and tvb loci segregate independently and recombine under the Mendelian second law of independent assortment. The dominant susceptibility es gene is expressed phenotypically when associated with the dominant susceptibility bs gene, but its expression is suppressed when associated with two doses of the recessive br gene. Genetic causes such as lack of penetration, recessive epistasis, and/or complementary intereaction between the tvb and tve genes have been discussed to account for the modified phenotypic expression of brbreses cells, i.e. resistance to subgroup E virus. Also, as reported previously, it was observed in this study that the tvb genes control the cellular response to subgroup D virus.

2-Deoxy-D-glucose uptake by chick embryo cells: a biochemical indicator of genetic susceptibility to RNA tumour viruses.

The enhanced glucose uptake by chick embryo cells as early as 72 h after infection with Rous sarcoma virus (RSV) was confirmed in this study to be an early indicator of cellular transformation. The glucose uptake of C/E BrL cells infected by various doses of BS-RSV of subgroup A showed that the relationship between the log-dose of virus and log-uptake was linear (slope, b1 = 1.30 +/- 0.14) when the ratio of the number of infectious virus particles to the number of cells in the culture was above 1:200. But infection of cultures with a relatively high dose of virus, for instance 10(3.5) focus forming units (f.f.u.) was ineffective for the measurement of cellular transformation using the criterion of glucose uptake, whereas a much lower dose such as 10(1.7) f.f.u. was sufficient to induce foci of transformed cells. We concluded therefore that the statistic of glucose uptake assay (GUA) measured at 72 p.i. is less sensitive than that of the focus count assay (FCA) measured after 10 days as a measure of assessing cellular transformation by RSV. Nevertheless, when the cultures were infected with a higher dose of virus (10(4.3) f.f.u. or more), the GUA could discriminate between the transformed (T) and non-transformed (NT) cultures. This was demonstrated in the two genetic crosses, line 7-2 X WC(F1) and line 7-2 C line. Embryo cultures of these two test-crosses were infected with viruses of subgroups A, B, C and D, and the T and NT phenotypes were ascertained. Also, on the basis of focus counts in the cultures the genetically resistant (R) and susceptible (S) phenotypes in response to various infections were determined. The T and NT phenotypes based on the GUA were compared with the S and R phenotypes, respectively, based on the FCA. It was found that in 47 of the 51 cultures, the phenotypic agreement was perfect, suggesting that glucose uptake by cells of embryo cultures exposed to RSV is a biochemical indicator of genetic susceptibility. The discordant results in 4 cultures are discussed in the light of present knowledge of cellular transformation by RSV.

Genetic susceptibility of chicken times quail hybrid embryos to avian RNA tumour viruses.

An attempt was made to hybridize the chicken (Gallus domesticus) male with Japanese quail (Coturnix coturnix japonica) female in order to study the genetic susceptibility of hybrid embryos to avian RNA tumour viruses of subgroups, A, B, D and E. In the hybrids the results supported the prevailing concept that susceptibility is dominant over resistance regardless of the dominant trait contributed by either parent. It was also observed that the Ie gene of the chicken was unable to suppress the 'quail-coded' susceptibility to subgroup E virus in the hybrid system, suggesting the lack of penetrance of the Ie gene. Despite the fact that some hybrids were resistant to viruses of subgroups B and D, they were susceptible to subgroup E virus, which was not expected on the basis of the concept that subgroup B-resistant cells cannot be E-susceptible. Also, the hybrids were susceptible to E virus regardless of gs antigen expression and presence of the Ie gene in the genome. This indicates that our earlier suggestion that the Ie gene is another expression of the gs antigen-determining gene is inconsistent.