An atypical clinicopathological manifestation of fowlpox virus associated with reticuloendotheliosis virus in commercial laying hen flocks in Brazil.

Fowlpox (FP) is a common epitheliotropic disease in chickens that is usually controlled by live attenuated vaccines. However, there have been some reports of outbreaks of FP in recent years, even in vaccinated flocks, presenting as atypical lesions and feathering abnormalities in chickens. These findings can be associated with fowlpox virus (FPV) with the reticuloendotheliosis virus (REV) integrated into its genome. In the present study, outbreaks of atypical FP were explored in vaccinated commercial laying hen flocks to determine the nature of the causative agent by histopathologic and molecular approaches. FPV and REV were detected and classified into subclade A1 of the genus Avipoxvirus and subtype 3 of REV (REV3), respectively. Additionally, heterogeneous populations of FPV with partial (containing only a remnant long terminal repeat-LTR) or total (all functional genes) integration of REV were identified by heterologous PCRs and detected considering reference integration sites. These results indicate the mechanism of chimeric genome FPV-REV associated with outbreaks and atypical clinicopathological manifestations in commercial laying hens for the first time in Brazil and in South America. In addition, this study demonstrates the emergence of REV integrated in the FPV genome in Brazilian chicken flocks.

Experimental infection of Attwater's/greater prairie chicken hybrids with the reticuloendotheliosis virus.

Reticuloendotheliosis virus (REV), a common pathogen of poultry, has been associated with runting and neoplasia in an endangered subspecies of grouse, the Attwater's prairie chicken. The pathogenesis of REV infection was examined in experimentally infected prairie chickens. Three groups of four Attwater's/greater prairie chicken hybrids were infected intravenously with varying doses (tissue culture infective dose [TCID50], 200, 1000, and 5000) of a prairie chicken-isolated REV. A fourth group of four birds was not infected. Blood was collected prior to infection, and at various times up to 37 wk following infection. Peripheral blood mononuclear cells were examined for integrated proviral DNA by a single-amplification polymerase chain reaction (PCR) and nested PCR of a region within the pol gene. The nested PCR identified REV proviral DNA in all REV-inoculated birds by 2 wk postinfection and confirmed chronic infection throughout the study. With the exception of a bird that died from bacterial pneumonia 8 wk postinfection, neoplasia, resembling that seen in naturally occurring infections, was observed in all birds, even those receiving as little as 200 TCID50 of virus.

Reticuloendotheliosis virus integration in the fowl poxvirus genome: not a recent event.

Integration of reticuloendotheliosis virus (REV) into the genome of fowl poxvirus (FPV) has been reported recently. With a view to determine whether this event had occurred in the past, we screened by polymerase chain reaction (PCR) for the presence of REV provirus in the DNAs of nine avian poxviruses, some of which had been lyophilized 50 yr ago. For REV, 5' long terminal repeat (LTR) and REV envelope sequences were amplified, whereas for FPV, the major envelope antigen gene and the region flanking REV sequences were amplified. In six of seven FPV strains examined, the specific PCR amplicons were obtained for both REV provirus and FPV sequences. One isolate in which presence of REV 5' LTR and envelope was not detected by PCR, a LTR remnant was detected by Southern hybridization. Interestingly, no REV sequence was detected in either canary poxvirus or pigeon poxvirus genome. These observations indicate that REV integration in the FPV genome is not a recent phenomenon but probably occurred prior to 1949.

Avian reticuloendotheliosis virus strain A and spleen necrosis virus do not infect human cells.

Spleen necrosis virus (SNV) and Reticuloendotheliosis virus strain A (REV-A) belong to the family of reticuloendotheliosis viruses and are 90% sequence related. SNV-derived retroviral vectors produced by the REV-A-based D17.2G packaging cell line were shown to infect human cells (H.-M. Koo, A. M. C. Brown, Y. Ron, and J. P. Dougherty, J. Virol. 65:4769-4776, 1991), while similar vectors produced by another SNV-based packaging cell line, DSH134G, are not infectious in human cells (reviewed by R. Dornburg, Gene Ther. 2:301-310, 1995). Here we describe a careful reevaluation of the infectivity of vectors produced from the most commonly used REV-A- or SNV-based packaging cells obtained from various sources with, among them, one batch of D17.2G packaging cells obtained from the American Type Culture Collection. None of these packaging cells produced vectors able to infect human cells. Thus, contrary to previously published data, we conclude that REV-based vectors are not infectious in human cells.

Nonreciprocal pseudotyping: murine leukemia virus proteins cannot efficiently package spleen necrosis virus-based vector RNA.

It has been documented that spleen necrosis virus (SNV) can package murine leukemia virus (MLV) RNA efficiently and propagate MLV vectors to the same titers as it propagates SNV-based vectors. Although the SNV packaging signal (E) and MLV packaging signal (Psi) have little sequence homology, similar double-hairpin RNA structures were predicted and supported by experimental evidence. To test whether SNV RNA can be packaged by MLV proteins, we modified an SNV vector to be expressed in an MLV-based murine helper cell line. Surprisingly, we found that MLV proteins could not support the replication of SNV vectors. The decrease in titer was approximately 2,000- to 20,000-fold in one round of retroviral replication. RNA analysis revealed that SNV RNA was not efficiently packaged by MLV proteins. RNA hybridization of the cellular and viral RNAs indicated that SNV RNA was packaged at least 25-fold less efficiently than MLV RNA, which was the sensitivity limit of the hybridization assay. The contrast between the MLV and SNV packaging specificity is striking. SNV proteins can recognize both SNV E and MLV Psi, but MLV can recognize only MLV Psi. This is the first demonstration of two retroviruses with nonreciprocal packaging specificities.

Retroviral insertional activation in a herpesvirus: transcriptional activation of US genes by an integrated long terminal repeat in a Marek's disease virus clone.

Insertional activation of host proto-oncogenes has been recognized as a basic mechanism by which nonacute retroviruses induce cancer. Our previous work has demonstrated that retroviruses can efficiently integrate into DNA virus genomes. Specifically, coinfection of cultured fibroblasts with a chicken herpesvirus, Marek's disease virus (MDV), and a chicken retrovirus results in frequent stable retroviral insertions into the herpesvirus genome. Such insertions could alter the expression of herpesvirus genes, possibly resulting in novel phenotypic properties. In this article, we report the characterization of a replication-competent clone of MDV with integrated retroviral sequences. This virus was isolated from a chicken following injection of fibroblasts coinfected with MDV and the retrovirus, reticuloendotheliosis virus. Transcripts originating from the reticuloendotheliosis virus long terminal repeat promoters were found to encode the adjoining MDV genes, SORF2, US1, and US10. This virus replicates well in culture but has an unusual phenotype in chickens, characterized by an attenuated virulence which produces no nerve lesions but, rather, severe thymic atrophy. While the causal relationship between the insertion and the observed phenotypes remains to be established, our data provide the first evidence of retroviral insertional activation of herpesvirus genes.

Reticuloendotheliosis viruses and derived vectors.

In most human gene therapy trials, the tools of gene delivery are retroviral vectors. All current vectors are derived from murine leukemia virus (MLV). Although this system is suitable for delivering a large variety of genes into different tissues, it also has its limitations and is not adequate for all potential applications of human gene therapy. Thus, attempts are underway in many laboratories to develop other gene delivery tools. Potential agents range from non-viral based gene delivery systems (eg liposomes) to other viral vectors such as those derived from adenoviruses, adeno-associated viruses, herpes simplex virus, and several other viruses. Furthermore, the development of other, non-MLV retroviral vector systems, including one derived from HIV, is in progress in several laboratories. In this article, reticuloendotheliosis viruses and their vector systems are reviewed, and their possible use in human gene therapy is discussed.

Pseudotyped REV/SRV retroviruses reveal restrictions to infection and host range within members of the same receptor interference group.

The reticuloendotheliosis virus (REV) group of retroviruses and type D simian retroviruses (SRV) belong to the same receptor interference group. The cellular receptor for these viruses has not yet been identified. In order to study the distribution of the receptor and to identify a receptor negative cell line, vector viral pseudotypes between REV and SRV were made. Using these viral pseudotypes, susceptibility to infection was examined in some rodent and marsupial cell lines. Infectivity assays with these envelope pseudotypes demonstrated that all cell types tested were resistant to infection. However, treatment of the rodent cells with the N-linked glycosylation inhibitor tunicamycin rendered most of the cells susceptible to infection. These results indicate that all the rodent cells tested express a nonfunctional receptor for viruses of the REV and SRV groups, which can be made functional by tunicamycin treatment. A difference in receptor host range among members of the same receptor interference group was observed which suggests that the REV/SRV receptor for this interference group might be differentially modified in different cell types. The studies also identified at least one cell line not expressing the REV/SRV receptor which should be useful for isolating the gene encoding the receptor.