A study of the relationship of reticuloendotheliosis virus to the avian leukosis-sarcoma complex of viruses

Reticuloendotheliosis virus (REV) was compared on biological and biochemical grounds with members of the avian leukosis-sarcoma complex of viruses (ALSV). Stocks of REV contained no virus which produced interference with any subgroup of ALSV. REV was unable to complement various strains of Rous sarcoma virus (RSV) as measured by the absence of broadening of host range, “helper” virus activity with the defective Bryan high titer strain of RSV, or the induction of release of RAV-60 from chick embryo cells containing the chick helper factor. Hence, no biological interaction has been detected between REV and the avian leukosis-sarcoma complex of viruses.The RNA structure of the REV was studied using glycerol velocity gradients and polyacrylamide gel electrophoresis. The RNA of REV was distinctly like that of RAV-49, a subgroup C avian leukosis virus. The native RNA of REV recovered after phenol extraction cosedimented with the RNA of RAV-49, and after heat denaturation, both RNAs showed the reduced sedimentation velocity and increased rate of migration in polyacrylamide gel which is characteristic of the tumor virus RNAs.Proteins of REV were examined on SDS discontinuous gel electrophoresis. Five major proteins were detected, two of which were glycoproteins. Coelectrophoresis of REV with avian sarcoma virus B₇₇ showed no polypeptides that were identical in electrophoretic mobilities. In addition, lactoperoxidase catalyzed iodination was employed to selectively label the surface proteins of REV. Two surface proteins were detected; these corresponded to the two viral glycoproteins.     

Radioimmunoassay for the envelope glycoprotein of subgroup E avian leukosis-sarcoma viruses.

The envelope glycoprotein of subgroup E avian leukosis viruses (gpE) was purified from Rous-associated virus type 0 (RAV-0) and used in double-antibody competition radioimmunoassays (RIA) and radioimmune precipitations (RIP). When embryo extracts from various inbred lines of chickens were tested, the results of assays for chick helper factor (chf) and RAV-0 were in complete phenotypic agreement with those of RIA for gpE. The expression of genes at the gs and gp loci varied among inbred lines. Normal line 15I₅ embryos contained gpE but not group-specific (gs) antigens, whereas extracts from virus-free lines 15₁ and 6₁ contained both classes of antigen. Line 15_B embryo extracts were negative for both gpE and gs antigen. Soluble gpE was found in sera of all chf-positive chickens from RAV-0-free lines. Sera from adult line 15_B birds were categorized as: (1) positive for RAV-0 but negative for antibody; (2) negative for RAV-0 but positive for gpE and antibody to gpE; and (3) negative for RAV-0, antibody to gpE, and gpE. Evidence of antibody production was completely correlated in RIP of iodinated gpE and serum neutralizations of subgroup E virus. Results of RIP and gpE RIA indicated that half of the sera from nonviremic line 15_B chickens contained antibodies to gpE as well as gpE.     

Feather pulp organ cultures for assessing host resistance to infection with avian leukosis-sarcoma viruses

The purpose of this study was to improve in vitro procedures for detecting cellular resistance to the avian leukosis-sarcoma group of viruses. Four feather pulp organ cultures (FPOC) were prepared from each chicken by placing pulp squeezed from feathers in wells of microtitre plates that contained culture medium. Two of the four FPOC were inoculated with Rous sarcoma virus (RSV) of subgroup A and 5 to 6 days later the fluids from all four cultures were assayed for virus by inoculating chicken embryo fibroblasts (CEF) and examining for development of foci of transformed cells. Prior to the second assay of culture fluids, quail cells transformed by envelope-defective RSV [R(-)Q cells] were added to some RSV-inoculated and uninocu-lated FPOC. The R(-)Q cells produce infectious RSV when infected with avian leukosis virus (ALV), and hence made it possible to detect ALV in FPOC. Status of host infection was also assessed by tests for virus neutralising antibody and the enzyme-linked immunosorbent assay for group specific viral antigen. In one experiment FPOC from chickens not exposed to ALV were susceptible to RSV throughout the 140-day test period. In contrast, FPOC from ALV-inoculated chickens were usually infected with ALV and were resistant to RSV. FPOC from chickens reared in contact with the inoculated group for 121 days were free of ALV and were unexpectedly resistant to RSV. Two other experiments supported the observation that genetically susceptible chickens acquire cellular resistance to RSV as a result of persistent or transient ALV-infection. In Cornell K strain chickens there was close agreement between cellular susceptibility based on tests on FPOC prepared prior to inoculation of chickens with ALV and for antibody following inoculation with ALV. A New Hampshire strain showed a high degree of genetic cellular resistance by these test procedures.     

A comparative study of the avian reticuloendotheliosis virus: relationship to murine leukemia virus and viruses of the avian sarcoma-leukosis complex.

Reticuloendotheliosis virus (REV) with several related viruses comprises a group of C-type, avian RNA tumor viruses which is distinct from the avian leukosis-sarcoma virus (ALSV) complex. The emphasis of the present investigation has been on the comparison of the properties of REV with those of a model mammalian RNA tumor virus, murine leukemia virus (MuLV), and with those of the ALSV.The structure and pathway of maturation of these viruses has been examined using electron microscopy. Conclusions derived from this work indicate that while the immature particles of REV can be morphologically distinguished from both MuLV and ALSV, the mature REV particle is very like that of MuLV and quite different from that of ALSV.The properties of the purified DNA-polymerase of these viruses were analyzed with the following significant findings: 1) Unfrozen, purified disrupted REV, but not purified REV-DNA-polymerase is enzymatically active using natural viral RNA as template-primer; 2) the DNA-polymerase copurifies with an RNase-H activity which probably resides on the same polypeptide; 3) the size of the DNA-polymerase-RNase-H complex is indistinguishable from that of the MuLV, a single polypeptide of 84,000 daltons; 4) the divalent cation preference of the REV-DNA polymerase, like that of MuLV, but unlike that of ALSV, is for Mn²⁺; and 5) serological cross-reaction between the DNA-polymerase of REV and ALSV could not be demonstrated.Apart from these structural and biochemical analogies, no direct relationship between REV and MuLV has been established. Infectivity of REV in two strains of mouse cells could not be demonstrated. Immunodiffusion tests for reaction of purified, disrupted REV with antisera specific for ALSV structural components and for the interspecies specific reaction characteristic of mammalian RNA tumor virus p 30 protein were uniformly negative. After consideration of all the available data, it seems that the REV must be considered a distinct group of avian RNA tumor viruses with significant structural similarities to mammalian viruses, but nonetheless differing antigenic determinants.     

Deciphering transmission dynamics and spillover of avian influenza viruses from avian species to swine populations globally

Virus Genes - Genome sequences of eleven avian influenza virus (AIV) subtypes have been reported in swine populations from seven countries until August 2020. To unravel the transmission dynamics...     

Murine xenotropic type C viruses. III. Phenotypic mixing with avian leukosis and sarcoma viruses.

Phenotypic mixing has been demonstrated between murine and avian type C viruses. Pseudotypes of Rous sarcoma virus (RSV) have been obtained which carry envelope determinants derived from xenotropic or ecotropic murine leukemia viruses (MuLV). Mouse sarcoma virus (MSV) pseudotypes have been constructed with envelope characteristics of subgroup C and E avian leukosis viruses. RSV pseudotypes produced with MuLV can infect mammalian cells, and MSV pseudotypes with avian leukosis virus infect chicken cells. The expression of transformation by these sarcoma genomes seems suppressed in certain of the heterologous hosts. Exogenous infection of mammalian cells by RSV(MuLV) results in not only transformation of the heterologous host but also the production of infectious progeny virus. The extent of this virus production appears determined by the ability of the helper virus to replicate in the host cell.     

A study of plaque formation with avian RNA tumor viruses.

Avian leukosis viruses of subgroups B, D, F, and one strain of reticuloendotheliosis virus (REV) are able to make plaques in several lines of chick embryo fibroblasts. Moreover, plaque formation with some strains of subgroup A is obtained only in chicken fibroblasts of $\text{C}\text{C}$ phenotype. One exception is RAV-3 which produces plaque in line 6 ($\text{C}\text{E}$) only. Several important steps for the reproducibility of the assay are described. Of all the vital stains tested, only neutral red incorporated into the agar overlay is capable of inducing the formation of plaques. Several lysosome stabilizers and labilizers are examined and their role in the mechanism of plaque formation is discussed.     

Endogenous leukosis viruses in the avian family Phasianidae.

Helper virus. activity for the defective Bryan high titer strain of Rous sarcoma virus (RSV) has been found in normal embryo fibroblast cultures of Ghighi, green, Mongolian, silver, and Swinhoe pheasants, and of Chinese quail and chukar. The helper viruses from Chinese quail and from Swinhoe pheasant were isolated from their respective RSV pseudotypes. Chinese quail, Ghighi, and golden pheasant cultures were also found to synthesize endogenous virus spontaneously. No such spontaneous production was seen with the other avian species investigated. The helper activity demonstrated in Chinese quail, chukar, Mongolian pheasant, and Swinhoe pheasant appears to specify envelope determinants which are different from those previously described. Viruses from Ghighi and silver pheasant have the G envelope, and those from green pheasant the F envelope specificity. The RSV pseudotype formed with Chinese quail virus shows a high plating efficiency on fibroblasts from mouse and European field vole. Chinese quail virus and Swinhoe pheasant virus also form plaques in chicken cells. Whether these pheasant viruses belong to established avian oncovirus groups, or whether they represent new ones, will have to be decided by immunological and biochemical studies now in progress.     

Helper viruses associated with avian acute leukemia viruses inhibit the cellular immune response

The cellular immune competence of lymphocytes obtained from birds infected with acute or chronic leukemia viruses was studied. The in vitro blastogenic response of thymus-derived lymphocytes (T cells) to the mitogens phytohemagglutinin (PHA) and connanavalin A (Con A) correlates with the ability of birds to elicit a cellular immune response (C. K. Naspitz and M. Richter, 1968, Prog. Allergy12, 1–85; P. Toivanen and A. Toivanen, 1973, J. Immunol.111, 1602–1603). The PHA response of splenic lymphocytes from birds infected with the avian acute leukemia viruses, myelocytomatosis virus strain 29 (MC29), avian erythroblastosis virus (AEV), avian myeloblastosis virus (AMV), or reticuloendotheliosis virus (REV-T), was completely suppressed within 1 week after virus infection of chickens by avian acute leukemia virusus. Spleen cells from chickens infected with the chronic leukemia viruses, Rous-associated viruses (RAVs) types 1 and 3, exhibited PHA responses similar to those of uninfected birds. In contrast, lymphocytes from RAV-2 infected birds had suppressed PHA responses. Acute leukemia viruses are replication-defective and are associated with replication-competent nontransforming helper viruses. When chickens were infected with helper viruses isolated from the acute leukemia virus stocks by endpoint dilution, the T-cell response to various T-cell mitogens was completely suppressed. The helper viruses, therefore, contribute to the immunosuppression which occurs during the development of avian acute leukemia. The rapid lethality of the acute leukemia viruses could be, in part, the result of the immunosuppressive activity of the associated helper viruses. Though all the acute leukemia viruses severely immunosuppress their hosts, the mechanism by which the immunosuppression was induced by these viruses differed. REV-T was the only acute leukemia virus which induced a population of splenic suppressor cells. All the viruses of the leukosis-sarcoma complex tested which had subgroup B specificity were immunosuppressive while those of subgroup A were not. These results suggest that immunosuppression by these viruses may be related to subgroup specificity.     

Polyethylene glycol-mediated infection with avian sarcoma viruses

Summary In this communication we report that fusion of viral and cellular membranes by polyethylene glycol is a convenient approach to overcoming genetically determined resistance to infection. Using this method, avian sarcoma virus-transformed mammalian cells have been produced which serve as useful model systems for the study of Rous sarcoma virus-specific tumor antigens.     

Pseudotypes of avian sarcoma viruses with the envelope properties of vesicular stomatitis virus.

Upon superinfection of cells producing Rous sarcoma virus (RSV) with temperature-sensitive mutants of vesicular stomatitis virus (VSV), two kinds of pseudotype viruses are produced: VSV genomes within particles bearing the envelope antigens of RSV, denoted VSV(RSV), and RSV genomes within particles bearing the envelope antigens of VSV, denoted RSV(VSV). The VSV(RSV) pseudotypes are recognized as the fraction of plaque-forming units resistant to neutralization by antiserum to VSV or, in the case of thermolabile envelope mutants of VSV, resistant to heat inactivation; they possess the host range restrictions of RSV and are neutralized by antisera specific to the RSV subgroup. The RSV(VSV) pseudotypes are recognized as the fraction of focus-forming units which transforms chick cells resistant to infection with the strain of RSV used. Both kinds of pseudotypes are produced concomitantly with VSV synthesis. VSV(RSV) particles comprise up to 12% of the VSV progeny titer and RSV(VSV) up to 1% of the RSV titer, but pseudotype fractions varied according to the VSV mutant used for superinfection. The proportions of pseudotypes in harvests of mixed infections are not reduced by filtration through 0.2-μm pore size filters to eliminate large aggregates of virus particles, and pseudotypes are not formed by mixing pure-grown RSV and VSV particles in vitro. VSV acts as a helper virus for BH-RSV(-), which is defective in envelope antigen, but not for αBH-RSV(-), which is also defective in RNA-directed DNA polymerase activity. The titer of BH-RSV(VSV) is enhanced by the presence of the avian leukosis helper virus, RAV-1, and more than 90% of this mixed pseudotype stock is neutralized by antiserum to either VSV or RAV-1, indicating that the RSV particles bear a mosaic of both VSV and RAV-1 envelope antigens. RSV(VSV) pseudotypes transform cells of four out of five mammalian species tested. Like RSV of subgroup D and B77, the focus-forming titer of RSV(VSV) assayed on mammalian cells is 1000-fold lower than on chick cells.     

Temperature-sensitive mutants of avian sarcoma viruses: genetic recombination between multiple or coordinate mutants and avian leukosis viruses.

Five coordinate temperature-sensitive mutants of avian sarcoma viruses which fail to transform or produce infectious progeny at 41° have been analyzed by genetic recombination. Four, namely LA334, 336, 338, and 343, carry multiple mutations. One of these mutations is always in the src gene affecting initiation and maintenance of transformation. The other mutations have not been mapped, but our data suggest that in LA338 there is no linkage of the second mutation with env, whereas in LA343 there is some linkage to env. LA336 has a second mutation affecting an early transient function in accordance with physiological data which have shown a thermolabile polymerase in this virus. The data on LA334 are in accord with previous studies which have indicated lesions in src and gag. For LA337 our data confirm the existence of single coordinate lesion segregating from env.     

Replication of avian influenza A viruses in mammals.

The recent appearance of an avian influenza A virus in seals suggests that viruses are transmitted from birds to mammals in nature. To examine this possibility, avian viruses of different antigenic subtypes were evaluated for their ability to replicate in three mammals-pigs, ferrets, and cats. In each of these mammals, avian strains replicated to high titers in the respiratory tract (10(5) to 10(7) 50% egg infective doses per ml of nasal wash), with peak titers at 2 to 4 days post-inoculation, similar to the pattern of human and other mammalian viruses in these animals. Most avian strains were recovered for 5 to 9 days post-inoculation. One avian H1N1 virus initially replicated poorly in pigs, but was adapted to this host and even transmitted to other pigs. Replication of the avian viruses occurred in the respiratory tracts of mammals, whereas, in birds, they replicate in the intestinal tract as well. The infected mammals had no significant disease signs and produced low levels of humoral antibodies; however, challenge experiments in ferrets indicated that they were immune. These studies suggest that influenza A viruses currently circulating in avian species represent a source of viruses capable of infecting mammals, thereby contributing to the influenza A antigenic pool from which new pandemic strains may originate.     

Serological evidence of human infections with avian influenza viruses

Summary Two hundred ninety-four subjects from Milan were tested for serum hemagglutination-inhibiting (HI) and neuraminidase-inhibiting (NI) antibodies to five avian influenza viruses. No HI antibodies were found in all the serum samples. On the contrary, NI antibodies to each strain were detected depending on the year of birth of the subjects.With 1 Figure     

Isolation of avian influenza viruses from two different transhemispheric migratory shorebird species in Australia

Summary. Shorebirds on their southerly migration from Siberia to Australia, may pass through Asian regions currently experiencing outbreaks of highly pathogenic H5N1 influenza. To test for the presence of avian influenza viruses in migratory shorebirds arriving in Australia during spring 2004, 173 cloacal swabs were collected from six species. Ten swabs were positive for influenza A, with H4N8 viruses detected in five red-necked stints and H11N9 viruses detected in five sharp-tailed sandpipers. No H5N1 viruses were detected. All isolated viruses were non-pathogenic in domestic chickens. These results further demonstrate the potential for migratory shorebirds to carry and potentially spread influenza viruses.     

Avian leukosis-sarcoma virus gene expression : Noncoordinate control of group-specific antigens in virus-negative avian cells

Cell-free extracts obtained from chicken embryos of inbred lines were used in double-antibody competition radioimmunoassays for the detection of avian leukosis-sarcoma virus polypeptides of molecular weight 27,000, 19,000, and 15,000 (p27, p19, and p15). Antigens were detectable at concentrations as low as 0.5–1 ng/mg of cell protein. Cells from line 100, which produces endogenous, Rous-associated virus, type O (RAV-O), competed for ¹²⁵I-labelled p27, p19, and p15 purified from avian myeloblastosis virus (AMV), whereas extracts from group-specific antigen-negative line 15 cells did not compete significantly for any of the three antigens tested. Extracts from the virusnegative line 6, which expressed group-specific antigen activity and chick-helper factor, competed with p27 and p19, but there was no detectable viral p15 in these cells. Analysis of viral polypeptides expressed in line 15× (line 6 × line 15)F₁ backcross embryos demonstrated the segregation of a dominant host gene for gs antigen reactivity, which was associated with the expression of both p27 and p19 in the absence of detectable p15. In contrast, two mammalian cell lines nonproductively transformed by Rous sarcoma virus expressed all three antigens at relative levels similar to those in intact virus.