Specificity of avian leukosis virus-induced hyperlipidemia

Rous-associated virus 7 (RAV-7) is a subgroup C avian leukosis virus which does not transform cells in vitro or carry an oncogene. When injected into 1-day-old hatched chicks, RAV-7 causes a low incidence of lymphoid leukosis after a latent period of several months. In contrast, infection of 10-day-old chicken embryos with RAV-7 leads to a disease syndrome characterized by stunting, obesity, atrophy of the bursa and the thymus, high triglyceride and cholesterol levels, reduced thyroxine levels, and increased insulin levels (Carter et al., Infect. Immun. 39:410-422, 1983; J.K. Carter and R.E. Smith, Infect. Immun. 40:795-805, 1983). Histopathological examination of tissues from affected chicks revealed an accumulation of lipid in the liver and an extensive infiltration of the thyroid and pancreas by lymphoblastoid cells. In the present investigation, the subgroup specificity of this syndrome was investigated. Other subgroup C avian leukosis viruses (transformation-defective B77, transformation-defective Prague C strain of Rous sarcoma virus, and RAV-49) caused stunting, infiltration of the thyroid and pancreas, increased liver weights, decreased thyroxine levels, and increased insulin levels, but they did not cause a uniform, profound increase in triglyceride and cholesterol levels. Avian leukosis viruses of subgroup A [myeloblastosis-associated virus 1 causing osteopetrosis [MAV-1(O)] and RAV-1], subgroup B [MAV-2(O), MAV-2 causing nephroblastoma [MAV-2(N)], and RAV-2], subgroup D (RAV-50), and subgroup F (ring-necked pheasant virus and RAV-61) did not cause a syndrome identical to that induced by RAV-7. All of the viruses examined induced some stunting and a reduction in thyroxine levels which correlated with the stunting. The two subgroup F viruses caused an infiltration of the thyroid which may have been secondary to severe lung involvement. We conclude that the RAV-7 syndrome is unique, particularly in the induction of a hyperlipidemia.     

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.     

Analysis of antigenic determinants of structural polypeptides of avian type C tumor viruses.

Radioimmunoassays were developed for the 19,000, 15,000, and 12,000 molecular weight polypeptides of avian myeloblastosis virus and for the 19,000 and 12,000 polypeptides of RAV-0, a subgroup E avian tumor virus. Each polypeptide was shown to possess both group- and type-specific antigenic determinants, in contrast to the 27,000 mol wt polypeptide, which contained only group-specific determinants. The corresponding low-molecular-weight polypeptides of subgroup A, B, and E viruses were shown to be immunologically indistinguishable. The findings that low-molecular-weight polypeptides of subgroup C and D viruses reacted very differently in immunoassays for the respective polypeptides of avian myeloblastosis virus or RAV-0 suggest that subgroups C and D may have evolved differently form subgroups A, B, and E.     

Antigenic relatedness between glycoproteins of human respiratory syncytial virus subgroups A and B: evaluation of the contributions of F and G glycoproteins to immunity.

The degree of antigenic relatedness between human respiratory syncytial virus (RSV) subgroups A and B was estimated from antibody responses induced in cotton rats by respiratory tract infection with RSV. Glycoprotein-specific enzyme-linked immunosorbent assays of antibody responses induced by RSV infection demonstrated that the F glycoproteins of subgroups A and B were antigenically closely related (relatedness, R approximately 50%), whereas the G glycoproteins were only distantly related (R approximately 5%). Intermediate levels of antigenic relatedness (R approximately 25%) were seen in neutralizing antibodies from cotton rats infected with RSV of the two subgroups. Immunity against the F glycoprotein of subgroup A, induced by vaccinia-A2-F, conferred a high level of protection which was of comparable magnitude against challenge by RSV of either subgroup. In comparison, immunity against the G glycoprotein of subgroup A, induced by vaccinia-A2-G, conferred less complete, but significant, protection. Importantly, in vaccinia-A2-G-immunized animals, suppression of homologous challenge virus replication was significantly greater (13-fold) than that observed for the heterologous virus.     

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.     

Nucleotide Sequences Derived from Pheasant DNA in the Genome of Recombinant Avian Leukosis Viruses with Subgroup F Specificity

Recombination between viral and cellular genes can give rise to new strains of retroviruses. For example, Rous-associated virus 61 (RAV-61) is a recombinant between the Bryan high-titer strain of Rous sarcoma virus (RSV) and normal pheasant DNA. Nucleic acid hybridization techniques were used to study the genome of RAV-61 and another RAV with subgroup F specificity (RAV-F) obtained by passage of RSV-RAV-0 in cells from a ring-necked pheasant embryo. The nucleotide sequences acquired by these two independent isolates of RAV-F that were not shared with the parental virus comprised 20 to 25% of the RAV-F genomes and were indistinguishable by nucleic acid hybridization. (In addition, RAV-F genomes had another set of nucleotide sequences that were homologous to some pheasant nucleotide sequences and also were present in the parental viruses.) A specific complementary DNA, containing only nucleotide sequences complementary to those acquired by RAV-61 through recombination, was prepared. These nucleotide sequences were pheasant derived and were not present in the genomes of reticuloendotheliosis viruses, pheasant viruses, and avian leukosis-sarcoma viruses of subgroups A, B, C, D, and E. They were partially endogenous, however, to avian DNA other than pheasant. The fraction of these nucleotide sequences present in other avian DNAs generally paralleled the genetic relatedness of these avian species to pheasants. However, there was a high degree of homology between these pheasant nucleotide sequences and related nucleotide sequences in the DNA of normal chickens as indicated by the identical melting profiles of the respective hybrids.     

Long terminal repeat (LTR) sequences, env, and a region near the 5'' LTR influence the pathogenic potential of recombinants between Rous-associated virus types 0 and 1.

A series of recombinants between Rous-associated virus type 0 (RAV-0), RAV-1, and a replication-competent avian leukosis virus vector (RCAN) have been tested for disease potential in day-old inoculated K28 chicks. RAV-0 is a benign virus, whereas RAV-1 and RCAN induce lymphoma and a low incidence of a variety of other neoplasms. The results of the oncogenicity tests indicate that (i) the long terminal repeat regions of RAV-1 and RCAN play a major role in disease potential, (ii) subgroup A envelope glycoproteins are associated with a two- to fourfold higher incidence of lymphoma than subgroup E glycoproteins, and (iii) certain combinations of 5' viral and env sequences cause osteopetrosis in a highly context-dependent manner. Long terminal repeat and env sequences appeared to influence lymphomogenic potential by determining the extent of bursal infection within the first 2 to 3 weeks of life. This would suggest that bursal but not postbursal stem cells are targets for avian leukosis virus-induced lymphomogenesis. The induction of neutralizing antibody had no obvious influence on the incidence of lymphoma.     

Suppression of Rous sarcoma virus-induced tumor formation by preinfection with viruses encoding src protein with novel N termini.

Two recovered avian sarcoma viruses (rASVs), rASV157 and rASV1702, encode src products which contain novel, nonmyristoylated N-terminal amino acids. These viruses transform chicken embryo fibroblasts and cause tumors in chicks. However, the tumors rASVs induce are small and regress within 2 weeks. To determine whether this regression results from weak tumorigenicity or from the active immunity of the host, we injected 1-week-old chicks with rASV and several days later injected the chicks with challenge virus of a different subgroup. Of the rASV1702-preinfected chicks challenged 5 days later with Rous sarcoma virus (RSV), 40% showed no subsequent tumor formation and 60% formed tumors which regressed within 1 week. The potency of this protective effect depended on the dosage of preinfection virus used and increased as the interval between preinfection and challenge infection was lengthened (when the interval was 9 days, none of the challenged chicks formed tumors). rASV157-preinfected chicks challenged with RSV after 9 days showed only partial protection: 42% formed tumors which regressed, whereas 58% formed tumors which continued to grow. Challenging rASV-preinfected chicks with Fujinami sarcoma virus or a RSV vector encoding the v-fps oncogene or polyomavirus middle T resulted in no suppression of tumor formation. Preinfection with src mutants or a RSV vector encoding polyomavirus middle T antigen, both of which induce slow-growing tumors, failed to elicit the protective effect. Finally, a novel N-terminal domain encoded by rASV1702 src was shown to be involved in but not sufficient for full protection. These data indicate that determinants on or induced by rASV157 and rASV1702 can elicit a potent protection against the tumorigenic potential of RSV-encoded p60v-src.     

Genetic analysis of the Rous sarcoma virus subgroup D env gene: mammal tropism correlates with temperature sensitivity of gp85.

Subgroup D avian sarcoma and leukosis viruses can penetrate a variety of mammalian cells in addition to cells from their natural host, chickens. Sequences derived from the gp85-coding domain within the env gene of a mammal-tropic subgroup D virus (Schmidt-Ruppin D strain of Rous sarcoma virus [SR-D RSV]) and a non-mammal-tropic subgroup B virus (Rous-associated virus type 2) were recombined to map genetic determinants that allow penetration of mammalian cells. The following conclusions were based on host range analysis of the recombinant viruses. (i) The determinants of gp85 that result in the mammal tropism phenotype of SR-D RSV are encoded within the 160 codons that lie 3' of codon 121 from the corresponding amino terminus of the gp85 protein. (ii) Small linear domains of the SR-D RSV gp85-coding domain placed in the subgroup B background did not yield viruses with titers equal to that of the subgroup D virus in a human cell line. (iii) Recombinant viruses that contained subgroup D sequences within the hr1 variable domain of gp85 showed modest-to-significant increases in infectivity on human cells relative to chicken cells. A recombinant virus that contained three fortuitous amino acid substitutions in the gp85-coding domain was found to penetrate the human cell line and give a titer similar to that of the subgroup D virus. In addition, we found that the subgroup D virus, the mutant virus, and recombinant viruses with an increased mammal tropism phenotype were unstable at 42 degrees C. These results suggest that the mammal tropism of the SR-D strain is not related to altered receptor specificity but rather to an unstable and fusogenic viral glycoprotein. A temperature sensitivity phenotype for infectivity of mammalian cells was also observed for another mammal-tropic avian retrovirus, the Bratislava 77 strain of RSV, a subgroup C virus, but was not seen for any other avian retrovirus tested, strengthening the correlation between mammal tropism and temperature sensitivity.     

芦花鸡中B亚群禽白血病病毒的分离与鉴定

通过接种DF-1细胞(C/E)系,从山东某地方品系芦花鸡的鸡群中分离到一株外源性白血病病毒(ALV)SDAU09C2。与GenBank中已发表的不同亚群鸡ALV参考株的囊膜蛋白gp85的氨基酸序列比较,表明该分离株与B亚群ALV(ALV-B)2个参考株的gp85的氨基酸同源性最高,均为92.5%;与A、C、D、E亚群ALV的gp85的氨基酸同源性仅在73.2%~87.9%之间;而与J亚群gp85的氨基酸同源性更低至30.3%~32.4%。这是我国地方品系鸡群中第一次分离和鉴定ALV-B及其gp85基因的报道。     

Component of Strain MC29 Avian Leukosis Virus with the Property of Defectiveness

Three clones of morphologically altered cells (L(-)MC29) of singular properties were isolated from MC29 (subgroup A) leukosis virus-infected chick embryo cells. Supernatant fluids from cultures of the cloned cells produced no transforming or interfering activity on chick embryo cells susceptible to known avian leukosis-sarcoma viruses. No virus associated with the cells was demonstrable by fluorescent-antibody staining or by electron microscopy. All L(-)MC29 clone cells were activated, however, by four strains of Rous-associated viruses (RAV) representative of A, B, C, and D subgroup avian leukosis viruses and by two strains of MC29 virus. Virus L(-)MC29 cells activated by superinfection with RAV-1 and RAV-2 was characterized by helper-dependent and helper-independent properties. These findings suggest that the strain MC29 leukosis virus, or a component thereof, possesses properties of defectiveness similar to those of the Bryan high-titer Rous sarcoma virus.     

Molecular basis of host range variation in avian retroviruses.

Previous genetic analysis has localized the region of the Rous sarcoma virus (RSV) env gene responsible for host range specificity to that encoding the middle one-third of gp85. To better understand the host range determinants, the relevant regions of the genomes of infectious molecular clones of the transformation-defective Prague strain of RSV, subgroup B (Pr-RSV-B) and Rous-associated virus 0 (RAV-0) (subgroup E) were sequenced and compared with the sequence of Pr-RSV-C. This comparative analysis identified two variable regions of low amino acid sequence homology flanked by highly conserved amino acid sequences. The first variable region (hr1) begins at base 5654 in the Pr-RSV-C sequence and encodes 32 amino acids. The second variable region (hr2) begins at base 5846 and encodes 27 amino acids. To test the role of the variable regions in host range specificity, we determined the sequence of this region of the env gene of NTRE-4, a recombinant virus between Pr-RSV-B and RAV-0 which exhibits an extended host range. This analysis revealed that the recombinant subgroup-encoding region of NTRE-4 is composed of 200 bases of RAV-0 sequence, including hr2, flanked by sequences which are otherwise of Pr-RSV-B origin. This study indicates that hr1 and hr2 are the domains of gp85 responsible for host range determination in avian retroviruses.     

Relationship of Reticuloendotheliosis Virus to the Avian Tumor Viruses: Nucleic Acid and Polypeptide Composition

The RNA content and polypeptide composition of reticuloendotheliosis virus (REV) was compared to that of C-type RNA tumor viruses. Two RNA species with approximate sedimentation values of 64S and 4S were observed after sucrose gradient centrifugation of RNA extracted from purified REV. The high-molecular-weight RNA species of REV sedimented slightly faster than that of the Bryan strain of Rous sarcoma virus (RSV). Although these characteristics were consistent with those of other C-type RNA tumor viruses, significant differences were observed when the polypeptide composition of REV was compared with that of RSV possessing envelope determinants of Rous-associated virus RAV-2 and RAV-3. Five polypeptides of which two were glycosylated were resolved by polyacrylamide gel electrophoresis. The major nonglycosylated polypeptide of REV did not comigrate with that of RSV (RAV-2)-RSV(RAV-3). The majority of the group-specific antigen reactivity resides in this major nonglycosylated polypeptide of avian tumor viruses and comigrates when proteins of several avian tumor viruses are subjected to coelectrophoresis. This difference in the migration of the major polypeptide of REV and RSV(RAV-2)-RSV(RAV-3) may explain the absence of avian tumor virus group-specific antigen in REV.     

Isolation and Identification of a Subgroup A Avian Leukosis Virus from Imported Meat-type Grand-parent Chickens

An exogenous avian leukosis virus (ALV) strain SDAU09C1 was isolated in DF-1 cells from one of 240 imported 1-day-old white meat-type grand parent breeder chicks. Inoculation of SDAU09C1 in ALV-free chickens induced antibody reactions specific to subgroup A or B. But gp85 amino acid sequence comparisons indicated that SDAU09C1 fell into subgroup A; it had homology of 88.8%-90.3% to 6 reference strains of subgroup A, much higher compared to other subgroups including subgroup B. This is the first report for A...     

Influence of env and long terminal repeat sequences on the tissue tropism of avian leukosis viruses.

Adsorption and penetration of retroviruses into eucaryotic cells is mediated by retroviral envelope glycoproteins interacting with host receptors. Recombinant avian leukosis viruses (ALVs) differing only in envelope determinants that interact with host receptors for subgroup A or E ALVs have been found to have unexpectedly distinctive patterns of tissue-specific replication. Recombinants of both subgroups were highly expressed in bursal lymphocytes as well as in cultured chicken embryo fibroblasts. In contrast, the subgroup A but not subgroup E host range allowed high levels of expression in skeletal muscle, while subgroup E but not subgroup A envelope glycoproteins permitted efficient replication in the thymus. A subgroup B virus (RAV-2), like the subgroup E viruses, demonstrated a distinct bursal and thymic tropism, further supporting the theory that genes encoding receptors for subgroup B and E viruses are allelic. The source of long terminal repeats (LTRs) or adjacent sequences also influenced tissue-specific replication, with the LTRs from endogenous virus RAV-0 supporting efficient replication in the bursa and thymus but not in skeletal muscle. These results indicate that ALV env and LTR regions are responsible for unexpectedly distinctive tissue tropisms.     

Anti-Rous sarcoma response of major histocompatibility (B) complex haplotypes B23, B24 and B30

Responses to Rous sarcoma virus (RSV) induced tumours were studied in UNH 105, a non-inbred line of New Hampshire chickens. Six single male matings encompassing a total of 50 dams produced 345 progeny which segregated for B complex genotypes B23/B23, B23/B24, B23/B30, B24/B24, B24/B30 and B30/B30. Six-week-old chicks were wingweb inoculated with a pseudotype of Bryan high titre Rous sarcoma virus, BH RSV (RAV-1). Tumours were scored for size six times over a 10-week period post-inoculation. Each chick was assigned a tumour profile index (TPI) as an indicator of immunological response. The number of days to death (DTD) was recorded for 148 chicks with terminal tumours. Genotypes B23/B23, B23/B24 and B23/B30, with TPIs of 1.8, 1.7 and 2.0 respectively, did not differ significantly from each other, suggesting dominance of response of B23 over B24 and B30 haplotypes. B24/B30 chicks with the highest TPI (3.4) and shortest DTD (34.6) were significantly different from B30/B30 (2.8; 41.6) but not from B24/B24 (3.1; 34.9) suggesting dominance of response of the B24 haplotype over B30 in the absence of B23.     

Replacement of the F and G proteins of respiratory syncytial virus (RSV) subgroup A with those of subgroup B generates chimeric live attenuated RSV subgroup B vaccine candidates

Human respiratory syncytial virus (RSV) exists as two antigenic subgroups, A and B, both of which should be represented in a vaccine. The F and G glycoproteins are the major neutralization and protective antigens, and the G protein in particular is highly divergent between the subgroups. The existing system for reverse genetics is based on the A2 strain of RSV subgroup A, and most efforts to develop a live attenuated RSV vaccine have focused on strain A2 or other subgroup A viruses. In the present study, the development of a live attenuated subgroup B component was expedited by the replacement of the F and G glycoproteins of recombinant A2 virus with their counterparts from the RSV subgroup B strain B1. This gene replacement was initially done for wild-type (wt) recombinant A2 virus to create a wt AB chimeric virus and then for a series of A2 derivatives which contain various combinations of A2-derived attenuating mutations located in genes other than F and G. The wt AB virus replicated in cell culture with an efficiency which was comparable to that of the wt A2 and B1 parents. AB viruses containing temperature-sensitive mutations in the A2 background exhibited levels of temperature sensitivity in vitro which were similar to those of A2 viruses bearing the same mutations. In chimpanzees, the replication of the wt AB chimera was intermediate between that of the A2 and B1 wt viruses and was accompanied by moderate rhinorrhea, as previously seen in this species. An AB chimeric virus, rABcp248/404/1030, which was constructed to contain a mixture of attenuating mutations derived from two different biologically attenuated A2 viruses, was highly attenuated in both the upper and lower respiratory tracts of chimpanzees. This attenuated AB chimeric virus was immunogenic and conferred a high level of resistance on chimpanzees to challenge with wt AB virus. The rABcp248/404/1030 chimeric virus is a promising vaccine candidate for RSV subgroup B and will be evaluated next in humans. Furthermore, these results suggest that additional attenuating mutations derived from strain A2 can be inserted into the A2 background of the recombinant chimeric AB virus as necessary to modify the attenuation phenotype in a reasonably predictable manner to achieve an optimal balance between attenuation and immunogenicity in a virus bearing the subgroup B antigenic determinants.     

Isolation and identification of a subgroup A avian leukosis virus from imported meat-type grand-parent chickens

An exogenous avian leukosis virus (ALV) strain SDAU09C1 was isolated in DF-1 cells from one of 240 imported 1-day-old white meat-type grand parent breeder chicks. Inoculation of SDAU09C1 in ALV-free chickens induced antibody reactions specific to subgroup A or B. But gp85 amino acid sequence comparisons indicated that SDAU09C1 fell into subgroup A; it had homology of 88.8%–90.3% to 6 reference strains of subgroup A, much higher compared to other subgroups including subgroup B. This is the first report for ALV of subgroup A isolated from imported breeders.     

RNA-directed DNA polymerase activity of reticuloendotheliosis virus: characterization of the endogenous and exogenous reactions.

Reticuloendotheliosis virus (REV) contains an endogenously instructed, RNA-directed DNA polymerase activity. Both the endogenous and exogenous DNA polymerase activities exhibited up to 10-fold greater activity at the optimum concentration of manganous ion (0.025 mM for exogenous; 0.25 mM for endogenous) than at any concentration of magnesium ion. Antiserum to the DNA polymerase of an REV group virus (spleen necrosis virus) inhibited both endogenous and exogenous DNA polymerase activity of REV, whereas antiserum to the Rous sarcoma virus (Rous-associated virus-0) [RSV(RAV-0)]DNA polymerase did not. The DNA product of the endogenous reaction is associated with the high-molecular-weight RNA of REV and anneals with REV RNA but not with RNA from Rous sarcoma virus.     

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.