Avian Vectors Research Articles

Contamination of fish products: risks and prevention.

The risks of contamination of finfish products with active pathogens largely depend on the type of product concerned and disposal methods of the importing country. Frozen fish used as bait or to feed high-value species present the greatest risk as vehicles of contamination because they are unprocessed. Freezing preserves viral- and some bacterial-pathogens, thus the use of such fish as bait can introduce those pathogens into natural waters. Conversely, processed fish, particularly fillets, which have been heat-treated or cooked, present the lowest risk. If fish are processed after importation, care must be taken to ensure effective waste disposal, with particular attention to the prevention of scavenging by avian vectors and drainage from landfills into natural waters. Liquid waste should be disinfected and disposed of well away from natural waters.

Approaches to food-based vaccines for domestic chickens

Domestic chickens were fed viral vaccines that were applied to the surface of food pellets. Responses were judged by the production of specific antibodies, and compared with the responses obtained when the same vaccines were given by conventional routes. Chickens responded similarly to commercial avian infectious encephalomyelitis vaccine given on food or by eyedrop when antibodies were measured by ELISA, and the vaccine virus spread by contact. Increasing the dose of oral vaccine tenfold gave a more rapid serological response but the levels of antibody were not increased. There was no serological response to commercial infectious laryngotracheitis virus vaccine given on food. An experimental avian adenovirus vaccine produced a serological response when given on food, but higher levels of antibody were produced in response to vaccination by eyedrop. The vaccine virus spread by contact. It was concluded that current avian infectious encephalomyelitis vaccines, and prospective recombinant vaccines based on avian adenovirus vectors, could be delivered on food.

Analysis of the subgroup A avian sarcoma and leukosis virus receptor: the 40-residue, cysteine-rich, low-density lipoprotein receptor repeat motif of Tva is sufficient to mediate viral entry

The genes encoding the receptor for subgroup A Rous sarcoma viruses (tva) were recently cloned from both chicken and quail cells (P. Bates, J. A. T. Young, and H. E. Varmus, Cell 74:1043-1051, 1993; J. A. T. Young, P. Bates, and H. E. Varmus, J. Virol. 67:1811-1816, 1993). Previous work suggested that only the extracellular domain of Tva interacts with the virus (P. Bates, J. A. T. Young, and H. E. Varmus, Cell 74:1043-1051, 1993). Tva is a small membrane-associated protein containing in its extracellular domain a 40-amino-acid region which is closely related to the low-density lipoprotein receptor (LDLR) repeat motif. To determine the region of the Tva extracellular domain responsible for viral receptor function, we created chimeric proteins containing various regions of the Tva extracellular domain fused with a murine CD8 membrane anchor. Analysis of these proteins demonstrates that any chimera containing the Tva LDLR repeat motif can specifically bind the envelope protein of subgroup A avian sarcoma and leukosis viruses. Furthermore, NIH 3T3 cell lines expressing these chimeric proteins were efficiently infected by subgroup A avian sarcoma and leukosis virus vectors. Our results demonstrate that the 40-residue-long LDLR repeat motif of Tva is responsible for viral receptor function.

V-jun cooperates with v-erbB to transform the thrombocytic/megakaryocytic lineage.

The transforming properties of v-jun, the viral counterpart of the transcription factor AP1, were investigated in avian hematopoietic cells. Two retroviruses, called JB and JBN, expressing both v-jun and v-erbB, were constructed using an avian erythroblastosis-based vector. We show that the cooperative action of both oncogenes allowed the virus to efficiently transform bone marrow cells. No such transformation was obtained with either oncogene alone. JB-transformed bone marrow cells expressed GATA-1, TAL-1, and histone H5, suggesting that they belong to the erythrocytic/thrombocytic lineage. (Thrombocytes are the avian homologues of mammal megakaryocytes.) Moreover, after induction with phorbol 12-myristate 13-acetate JB-transformed bone marrow cells began to differentiate and synthesized high levels of platelet glycoproteins, indicating that they were of thrombocytic origin. These results were confirmed by c-ets1 analysis since this transcription factor, specifically found in cells with megakaryocytic but not erythrocytic features, was clearly detected in these cells.

Where are we now with vaccines against AIDS?

Extensive studies in experimental animal models and phase I and II clinical trials in humans provide some grounds for optimism about developing a vaccine for AIDS. Over 200 monkeys have been protected by simple inactivated vaccines against infection with a lethal challenge dose of simian immunodeficiency virus (SIV). Passive transfer of the antibody to SIV has been shown to prevent infection. Recent vaccination with attenuated, live SIV protected against a challenge with 1000 monkey infectious doses of virus. Studies in chimpanzees have been limited to the unrepresentative IIIB strain of HIV-1. Purified recombinant envelope protein either as the gp 120 surface unit or as the entire gp 160 protein has protected a proportion of chimpanzees against infection. Several experimental immunogens have been tested in phase I and II clinical trials in human volunteers. 5 different recombinant HIV envelope subunits, live recombinant vaccinia virus expressing HIV envelope, and synthetic peptides or virus-like particles derived from yeast, but expressing HIV core proteins, have been used for vaccines that have proved safe. However, large quantities of the subunit HIV antigens are required, and the humoral immune responses are short-lived. Trials with the live recombinant vaccinia virus expressing HIV envelope represent the first use of live recombinant virus in humans. Further studies are under way in France with an avian poxvirus vector, which is host restricted and therefore potentially safer. STudies with combinations of live recombinant virus vaccines followed by immunization with purified subunits indicate that this approach may stimulate both cellular immunity and neutralizing antibodies at higher concentrations than previously observed. The WHO has identified sites in Brazil, Rwanda, Thailand, and Uganda for large scale trials of the efficacy of AIDS vaccines that will probably begin within 5 years.

Poxvirus vectors: cytoplasmic expression of transferred genes

During the past year there have been a number of advances in poxvirus vector design and application. These include improvements in gene expression and a direct method of inserting DNA into the poxvirus genome. Alternative methods of selecting recombinant viruses have been developed, and attenuated and non-replicating vaccinia virus and avian poxvirus vectors are now being used successfully. Field trials of an oral, wild-life rabies vaccine, and phase 1 testing of human vaccines derived from vaccinia virus are in progress. This review is an updated and modified version of a review first published in Current Opinion in Biotechnology 1992, 3:533-539.

Vaccinia and other poxvirus expression vectors.

Over the past year improvements have been made in recombinant vaccinia virus gene expression and a new method for inserting DNA into the poxvirus genome has been developed, along with alternative methods for selecting recombinant viruses. Attenuated and non-replicating vaccinia virus and avian poxvirus vectors are now being used successfully. Field trials of an oral, wild-life rabies vaccine and phase 1 testing of human vaccines derived from vaccinia virus are underway.

In situ expression of helper-free avian leukosis virus (ALV)-based retrovirus vectors in early chick embryos.

Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.

Mutations within the 5' half of the avian retrovirus MC29 v-myc gene alter or abolish transformation of chicken embryo fibroblasts and macrophages.

Avian myelocytomatosis virus MC29 induces a wide variety of neoplastic diseases in infected birds and transforms cells of the macrophage lineage as well as fibroblasts and epithelial cells. A biological and biochemical analysis, carried out on a series of in-frame insertion and deletion mutations within the gag-myc gene of MC29, revealed several mutations within the 5' portion of the v-myc gene that encode proteins either completely defective for transformation or compromised in their ability to transform chicken embryo fibroblasts but not macrophages. Mutations within the 3' end of the v-myc gene which disrupt sequences encoding the basic/helix-loop-helix region were defective for transformation of both fibroblasts and macrophages. Eight variants were cloned into the replication-competent avian expression vector RCAS. Analysis of cells infected with transformation-defective, replication-competent viruses confirmed the expression of functionally defective Myc proteins. Further, expression of the transformation defective variant dl91-137 in chicken fibroblasts inhibited subsequent transformation by wild-type MC29. The results reported herein support the hypothesis that Myc proteins function as regulators of transcription in a variety of cell types and clearly point out the necessity of putative regulatory domains within the amino-terminal half of the Myc protein.

Search for the origin of Multiple Sclerosis by first identifying the vector

By intensive study of environmental conditions during the final stages in an active cluster area, an attempt is made to crack the enigmatic deadlock concerning the origin and etiological cause of Multiple Sclerosis (MS). Previous investigations of other cluster areas have emphasized the probability that the outbreaks of MS were most likely caused by an unknown exogenous environmental agent, but unfortunately, the studies were conducted several years after the epidemics had occurred, making it virtually impossible to observe changing environmental conditions. This study attempts to overcome such defects, and in the process identifies not only the probable exogenous viral agent(s), but the here-to-fore unknown avian vectors as well.

Gene Expression from Heterologous Promoters in a Replication-Defective Avian Retrovirus Vector in Quail Cells

Avian retrovirus vectors, with potential for use in avian transformation, were constructed to evaluate the relative efficiency of promoters placed internal to the viral long terminal repeats (LTR). The vectors are replication-defective reticuloendotheliosis plasmids that contain the neomycin phosphotransferase gene under control of the 5′ LTR and an internal promoter that directs expression of the chloramphenicol acetyltransferase gene. The internal promoters were the SV40 early, the mouse metallothionein I, and the human cytomegalovirus immediate early (HCMV-IE) promoters. Under transient conditions in QT6 cells, the HCMV-IE promoter construct was by far the strongest. However, expression dropped greatly from the HCMV-IE promoter after integration into the quail cell genome. Evidence suggests that the HCMV-IE promoter is selectively suppressed by methylation after stable transfection but not after infection.

Survival of early preimplantation porcine embryos after co-culture with cells producing an avian retrovirus

To determine the relative survival of porcine embryos after co-culture with cells producing an avian retrovirus, four-cell stage embryos were obtained from sows following synchronization with altrenogest and superovulation with gonadotropins. These embryos were randomly assigned to the following treatments: no manipulation (zona-intact); zona removed with acidified Tyrode's solution (zona-free); and zona removed followed by co-culture with D-17 canine cells producing an avian retrovirus vector derived from spleen necrosis virus (zona-free + co-culture). The survival rates of four-cell stage embryos to morulae or early blastocysts during a 48-h culture period were 93.3, 80.0 and 57.7% in zona-intact, zona-free and zona-free + co-culture groups, respectively. Following embryo transfer, the development of embryos to fetuses at six weeks of gestation was 37.5, 30.0 and 11.7% in zona-intact, zona-free and zona-free + co-culture groups. These results indicate that early preimplantation porcine embryos can develop to apparently normal fetuses following co-culture with cells producing a retrovirus, and the feasibility of this method for retrovirus-mediated gene transfer in pigs was demonstrated.

Avian trypanosomes as models of hemoflagellate evolution

In the 15 years since the last review on avian trypanosomes 1, there has been a steady accrual of information on the distribution and dynamics of trypanosome infections in wild bird populations. Recent immunological and biochemical studies provide evidence that several trypanosome species can parasitize an ecological guild of host species but the relative roles of avian host phylogeny and vector ecology remain unanswered. In this article, Victor Apanius reviews the habitat preferences and behavior of trypanosomes within the avian host and attempts to draw similarities in the strategy employed by the parasite for persistence in different vertebrate classes. Next, the question of host specificity is raised and recent evidence on the subject is examined with an eye toward understanding the distribution of trypanosome species in host communities.

Ectopic expression of the erythrocyte band 3 anion exchange protein, using a new avian retrovirus vector

A retrovirus vector was constructed from the genome of avian erythroblastosis virus ES4. The v-erbA sequences of avian erythroblastosis virus were replaced by those coding for neomycin phosphotransferase, creating a gag-neo fusion protein which provides G418 resistance as a selectable marker. The v-erbB sequences following the splice acceptor were replaced by a cloning linker allowing insertion of foreign genes. The vector has been tested in conjunction with several helper viruses for the transmission of G418 resistance, titer, stability, transcription, and the transduction and expression of foreign genes in both chicken embryo fibroblasts and the QT6 quail cell line. The results show that the vector is capable of producing high titers of Neor virus from stably integrated proviruses. These proviruses express a balanced ratio of genome length to spliced transcripts which are efficiently translated into protein. Using the Escherichia coli beta-galactosidase gene cloned into the vector as a test construct, expression of enzyme activity could be detected in 90 to 95% of transfected target cells and in 80 to 85% of subsequently infected cells. In addition, a cDNA encoding the avian erythrocyte band 3 anion exchange protein has been expressed from the vector in both chicken embryo fibroblasts and QT6 cells and appears to function as an active, plasma membrane-based anion transporter. The ectopic expression of band 3 protein provides a visual marker for vector function in these cells.

Characterization of Chicken c-ski Oncogene Products Expressed by Retrovirus Vectors

We constructed replication-competent avian retrovirus vectors that contain two of the three known types of chicken c-ski cDNAs and a third vector that contains a truncated c-ski cDNA. We developed antisera that recognize the c-ski proteins made by the three transforming c-ski viruses. All three proteins (apparent molecular masses, 50, 60, and 90 kilodaltons) are localized primarily in the nucleus. The proteins are differentially phosphorylated; immunofluorescence also suggests that there are differences in subnuclear localization of the c-ski proteins and that c-ski protein is associated with condensed chromatin in dividing cells.

Characterization of chicken c-ski oncogene products expressed by retrovirus vectors.

We constructed replication-competent avian retrovirus vectors that contain two of the three known types of chicken c-ski cDNAs and a third vector that contains a truncated c-ski cDNA. We developed antisera that recognize the c-ski proteins made by the three transforming c-ski viruses. All three proteins (apparent molecular masses, 50, 60, and 90 kilodaltons) are localized primarily in the nucleus. The proteins are differentially phosphorylated; immunofluorescence also suggests that there are differences in subnuclear localization of the c-ski proteins and that c-ski protein is associated with condensed chromatin in dividing cells.

Isolation and characterization of fra-2, an additional member of the fos gene family.

Antiserum raised against a Fos peptide (amino acids Lys-127 to Arg-140 of chicken c-Fos) recognizes a 46-kDa Fos-related protein in cell lysates of growth-stimulated chicken embryo fibroblasts. Induction of the 46-kDa protein is transient but is slightly prolonged relative to c-Fos following growth stimulation. Using a mixed oligonucleotide probe encoding the peptide antigen, we have cloned the chicken genomic locus that encodes this protein and have determined its gene structure. This locus consists of four exons, each of which has some homology with the corresponding exons of the chicken c-fos gene, and it expresses a 6-kilobase mRNA after growth stimulation. The deduced amino acid sequence of the gene (323 amino acids) contains a "leucine zipper" and includes five distinct regions that exhibit strong sequence homology to the recently identified fos-related antigens Fra-1 (rat) and FosB (mouse) as well as c-Fos. Since the other regions of the gene have little homology to any of these three proteins, this gene was named "fra-2." When the fra-2 gene was overexpressed by an avian retrovirus vector, it caused transformation of chicken embryo fibroblasts. The fra-2 gene product formed a complex in cells with the c-Jun protein, indicating that c-fos and fra-2 share biological and biochemical functions.

Helper-independent retrovirus vectors with Rous-associated virus type O long terminal repeats

We have constructed nonpermuted replication-competent avian retrovirus vectors that derive from Rous sarcoma virus (S. H. Hughes, J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, J. Virol. 61:3004-3012, 1987). We describe here the construction and properties of corresponding vectors in which the long terminal repeats (LTRs) of the parental virus have been replaced by the LTRs of the endogenous chicken virus Rous-associated virus type O. The Rous-associated virus type O LTR vectors replicated approximately 1/10 as well as the parental vectors and expressed a test gene, chloramphenicol acetyltransferase, approximately 1/30 to 1/50 as well.

PNEUMONITIS WITH ALVEOLAR-CAPILLARY BLOCK IN A CATTLE RANCHER EXPOSED TO EPIZOOTIC BOVINE ABORTION.

AGENTS of the psittacosis-lymphogranuloma-venereum-trachoma group (bedsoniae) have been isolated in cases of pneumonitis in both animals and man, and transmission of psittacosis and ornithosis to man by avian vectors is now well recognized. Sporadic outbreaks of pneumonitis due to so-called of bedsoniae have occurred in San Francisco,1 the bayou region of Louisiana2 3 4 5 6 7 and Chicago.8 In these cases no animal reservoir of the infection could be directly incriminated. Cases of human infection with nonavian animal strains of bedsoniae have rarely been described. Two isolated cases of laboratory infection with the bedsonia strains responsible for bovine encephalomyelitis9 and enzootic abortion . . .