Discovery of an expanded set of avian leukosis subgroup E proviruses in chickens using Vermillion, a novel sequence capture and analysis pipeline [corrected].

Transposable elements (TEs), such as endogenous retroviruses (ERVs), are common in the genomes of vertebrates. ERVs result from retroviral infections of germ-line cells, and once integrated into host DNA they become part of the host's heritable genetic material. ERVs have been ascribed positive effects on host physiology such as the generation of novel, adaptive genetic variation and resistance to infection, as well as negative effects as agents of tumorigenesis and disease. The avian leukosis virus subgroup E family (ALVE) of endogenous viruses of chickens has been used as a model system for studying the effects of ERVs on host physiology, and approximately 30 distinct ALVE proviruses have been described in the Gallus gallus genome. In this report we describe the development of a software tool, which we call Vermillion, and the use of this tool in combination with targeted next-generation sequencing (NGS) to increase the number of known proviruses belonging to the ALVE family of ERVs in the chicken genome by 4-fold, including expanding the number of known ALVE elements on chromosome 1 (Gga1) from the current 9 to a total of 40. Although we focused on the discovery of ALVE elements in chickens, with appropriate selection of target sequences Vermillion can be used to develop profiles of other families of ERVs and TEs in chickens as well as in species other than the chicken.

Novel avian leukosis virus-related endogenous proviruses from layer chickens: characterization and development of locus-specific assays.

During the course of evolution, vertebrate genomes have been invaded and colonized by retroviruses. In humans, for example, endogenous retroviruses (long terminal repeat elements) occupy roughly twice as much sequence space as essential genes. There are numerous reports in the literature implicating endogenous proviruses in the modulation of host physiology. The fact that many of these host-virus interactions take place in a proviral locus-specific manner speaks to the need for rapid assays for element profiling. This report deals with the identification of novel elements belonging to a family of endogenous retroviruses, designated ALVE, that reside in the genome of the chicken and that are closely related to exogenous avian leukosis viruses. The study of ALVE elements in the chicken genome serves as a model system for understanding the interplay between endogenous viruses and their vertebrate hosts in general, including humans. In this report, we present locus-specific, diagnostic PCR-based assays for 2 novel ALVE elements. In addition, we characterize the proviral structures and examine the genomic environments of both novel elements along with a previously described element known as ALVE-NSAC-3.

On the ubiquity and phylogeny of Wolbachia in lice.

Wolbachia are intracellular bacteria that occur in an estimated 20% of arthropod species. They are of broad interest because they profoundly affect the reproductive fitness of diverse host taxa. Here we document the apparent ubiquity and diversity of Wolbachia in the insect orders Anoplura (sucking lice) and Mallophaga (chewing lice), by detecting single or multiple infections in each of 25 tested populations of lice, representing 19 species from 15 genera spanning eight taxonomic families. Phylogenetic analyses indicate a high diversity of Wolbachia in lice, as evidenced by the identification of 39 unique strains. Some of these strains are apparently unique to lice, whereas others are similar to strains that infect other insect taxa. Wolbachia are transmitted from infected females to their offspring via egg cytoplasm, such that similar species of lice are predicted to have similar strains of Wolbachia. This predicted pattern is not supported in the current study and may reflect multiple events of recent horizontal transmission between host species. At present, there is no known mechanism that would allow for this latter mode of transmission to and within species of lice.

A comparative map of bovine chromosome 19 based on a combination of mapping on a bacterial artificial chromosome scaffold map, a whole genome radiation hybrid panel and the human draft sequence.

We have constructed a medium density physical map of bovine chromosome 19 using a combination of mapping loci on both a bovine bacterial artificial chromosome (BAC) scaffold map and a whole genome radiation hybrid (WGRH) panel. The resulting map contains 70 loci spanning the length of bovine chromosome 19. Three contiguous groups of BACs were identified on the basis of multiple loci mapping to individual BAC clones. Bovine chromosome 19 was found in this study to be comprised almost entirely from regions of human chromosome 17, with a small region putatively assigned to human chromosome 10. Fourteen breakpoints between the bovine and human chromosomes were detected, with a possibility of five more based on ordering of the WGRH map.

Characterization of four endogenous viral genes in semi-congenic lines of meat chickens.

Restriction fragment length polymorphism analyses were used to examine endogenous viral genes (ev genes or ALVE genes) of the avian leukosis viral (ALV) family in semi-congenic lines of meat chickens. The Generation 6 lines examined in this study were semi-congenic in that each contained birds with either zero or with one ALVE gene in hemizygous state plus some solitary long terminal repeat (LTR) elements. Using four restriction enzymes on chicken genomic DNA and two probes, one representing the entire ALV retroviral genome and one with only a small part plus the LTR, four ALVE genes were characterized. Each seemed to be complete with no detectable deletions. None appeared to be similar to known ALVE genes of White Leghorns, whereas two of the four may be the same as ALVE genes reported by others in White Plymouth Rock chickens.

Sequence and phylogenetic analysis of the SNF4/AMPK gamma subunit gene from Drosophila melanogaster.

To optimize gene expression under different environmental conditions, many organisms have evolved systems which can quickly up- and down-regulate the activity of other genes. Recently, the SNF1 kinase complex from yeast and the AMP-activated protein kinase complex from mammals have been shown to represent homologous metabolic sensors that are key to regulating energy levels under times of metabolic stress. Using heterologous probing, we have cloned the Drosophila melanogaster homologue of SNF4, the noncatalytic effector subunit from this kinase complex. A sequence corresponding to the partial genomic sequence as well as the full-length cDNA was obtained, and shows that the D. melanogaster SNF4 is encoded in a 1944-bp cDNA representing a protein of 648 amino acids (aa). Southern analysis of Drosophila genomic DNA in concert with a survey of mammalian SNF4 ESTs indicates that in metazoans, SNF4 is a duplicated gene, and possibly even a larger gene family. We propose that one gene copy codes for a short (330 aa) protein, whereas the second locus codes for a longer version (<410 aa) that is extended at the carboxy terminus, as typified by the Drosophila homologue presented here. Phylogenetic analysis of yeast, invertebrate, and multiple mammalian isoforms of SNF4 shows that the gene duplication likely occurred early in the metazoan lineage, as the protein products of the different loci are relatively divergent. When the phylogeny was extended beyond the SNF4 gene family, SNF4 shares sequence similarity with other cystathionine-beta-synthase domain-containing proteins, including IMP dehydrogenase and a variety of uncharacterized Methanococcus proteins.

Locus-specific diagnostic tests for endogenous avian leukosis-type viral loci in chickens.

The genome of the chicken, Gallus gallus, contains endogenous proviral elements (ALVE elements or ev genes) that display a high degree of similarity to the Avian Leukosis class of retroviruses. The ALVE proviruses are known to modulate physiological processes of the host birds. Different ALVE elements retain variable portions of the complete, prototype viral genome, and each provirus resides in its own specific location within the host genome. Thus, each ALVE element has its own particular potential to modulate host physiology depending on the nature of its integration site, the completeness of the proviral genome, and the level of expression of the locus. It is important, therefore, to be able to establish the ALVE element profiles of chickens quickly and accurately, both in the laboratory and in a commercial setting. The current method of choice for simple, quick, and accurate typing is the polymerase chain reaction (PCR). This paper reviews the present status of PCR typing of ALVE proviruses and lists the assay protocols for 19 different elements. In addition, it compares the insertion sites of these elements in an effort to identify common motifs at ALVE integration sites.

A deleted retroviral insertion at the ev21-K complex locus in Indonesian chickens.

Very poor feather development has been observed in chickens of the Nunukan strain, originating from Indonesia. The wing of the newly hatched chick does not show any primary or covert feathers; this phenotype will be referred to as very-late feathering (VLF). As adults, chickens are feathered but tail feathers are short and fragile. An experimental population was set up at the National Institute of Agronomic Research (INRA), Jouy-en-Josas, from one Nunukan male and four Nunukan females. Preliminary observations did not support the hypothesis of a sex-linked dominant mode of inheritance for the VLF phenotype. A restriction fragment length polymorphism (RFLP) study using five restriction enzymes and two probes, RAV-2 and endogenous virus (ev) ev21-int specific for the endogenous viral locus ALVE21, showed the presence of the expected 3' junction fragments for the ev21 occupied site but failed to reveal the expected 5' junction fragments for ev21 in Nunukan chickens. The unoccupied site corresponded to the ev21 unoccupied repeat (UR) of type a (URa). A deletion in the 5' region of the provirus and of the insertion site was indicated by the RFLP analysis and confirmed by a PCR study. Primers were designed in order to amplify a 5' junction fragment specific to the modified ev21 found in the Nunukan chickens. The sequence of this amplified product showed that the deletion started 652 bp upstream of the insertion site of ev21 and ended within the pol gene of the viral genome. This deletion represents a new allele, OSD, at the ev21 insertion site (locus ALVE21), that appears insufficient to produce a complete virus. Current data do not show a clear causal relationship between OSD and the VLF phenotype. The presence of OSD may be required but is not in itself sufficient to obtain the VLF phenotype. The genetic relationships between OSD and the altered feathering phenotype of Nunukan chickens will be investigated further in families segregating for the VLF phenotype, using the locus-specific PCR test developed as part of this study.

Long range-inverse PCR (LR-IPCR): extending the useful range of inverse PCR.

The inverse PCR technique (IPCR) has proven to be very useful for the amplification of uncharacterized stretches of DNA upstream or downstream of regions that have already been cloned and sequenced. In practice, however, chromosome walking using standard IPCR is often a slow, repetitive process because only small DNA fragments are effectively amplified. The development of long and accurate PCR methodology has greatly expanded the range of DNA fragment sizes that is amenable to amplification by conventional PCR. We reasoned that combining long range PCR with IPCR would also extend the useful range of the IPCR technique. In this paper we demonstrate the utility of the hybrid, long range-inverse PCR (LR-IPCR) technique by generating clones containing long stretches of DNA flanking endogenous chicken proviral elements.

Characterization of eight endogenous viral (ev) genes of meat chickens in semi-congenic lines.

Restriction fragment length polymorphism analysis was conducted for a set of eight different meat chicken-derived endogenous viral genes (ev genes) of the avian leukosis viral (ALV) family. Each viral element was first isolated into a separate single-element line by selective breeding. Genomic DNA from the founder male for each semi-congenic, single-element line was digested with each of four restriction enzymes, and the resulting Southern blots were each hybridized with up to four probes representing different portions of the ALV retroviral genome. Among the eight elements, there was one that represents the broiler equivalent of locus ev3 of White Leghorn chickens. A second broiler element showed a SacI-specific junction fragment similar to that of ev8. The remainder appeared to be different from any of the 21 ev genes previously described for White Leghorn chickens. Four of the eight elements examined were essentially complete, but the rest have sustained internal deletions.

A rapid PCR-based test for the endogenous viral element ev3 of chickens.

A short fragment of chicken genomic DNA encompassing the insertion site of the endogenous avian leucosis viral element ev3 was isolated using the inverse polymerase chain reaction (inverse PCR) technique. The nucleotide sequence of the unoccupied site was used to design PCR primers that can be used to unambiguously determine the genetic status of any chicken, with respect to ev3. Screening of a small number of individuals from exotic breeds of chickens suggested that the frequency of ev3 is highly variable. The ev3 integration site shows a high degree of sequence homology with the macrophage-specific tyrosine kinase gene, bmk, in mice.

Influence of the alv6 recombinant avian leukosis virus transgene on production traits and infection with avian tumor viruses in chickens.

The biological costs of the alv6 recombinant transgene that in chickens induces dominant resistance to the subgroup A avian leukosis virus (ALV), in terms of effects on production traits, were studied. Four generations of White Leghorn chickens of Line TR, segregating for alv6 but free of endogenous viral genes, as well as two generations of crosses between TR and Ottawa Line WG (WGTR) were tested under a specific-pathogen-free environment. In the birds studied, the transgene appeared unchanged compared to the original alv6: No major changes in alv6 DNA were detected by restriction analysis, the transgene did not express the group-specific antigen of ALV, and its presence was associated with absence of immune response to ALV. In most test years, and both TR and WGTR genomic backgrounds, alv6 was associated with delayed sexual maturity by 4 to 6 d, reduced egg production to 497 d of age by 20 to 46 eggs, and a 3.6 to 15% decline in egg production rate. No consistent effects on other traits, including mortality, were detected. When inoculated with the AC-1 isolate of Marek's disease virus in a separate experiment, TR birds with alv6 had a significantly lower body weight gain to 10 d of age than their sibs without the transgene. Thus, transgenesis has biological costs that have to be assessed against desirable effects of transgenes.

A novel molecular fingerprint probe based on the endogenous avian retroviral element (EAV) of chickens.

We have developed a novel molecular probe that is useful for DNA fingerprint analysis in chickens. The probe is based on the middle-repetitive, chicken endogenous retroviral (EAV) element. It consists of 1503 bp of the 3' portion of the EAV element, extending from the down-stream end of the envelope gene to the beginning of the downstream long terminal repeat (LTR). Unlike other probes that are currently in use for fingerprint analysis with chicken DNA, the EAV-based probe works well at normal levels of stringency, and with standard hybridization buffers. Digestion of chicken genomic DNA with a variety of restriction enzymes routinely yields up to 30 resolvable bands per bird in the 500 bp to 20 kbp range. In order to test the efficacy of the EAV-based fingerprint probe, we have used it to estimate the degree of inbreeding in the inbred WG strain of White Leghorns. We find that the estimates derived with the EAV probe are very similar to those reported previously for the WG strain. These results suggest that molecular probes based on endogenous retroviruses and other middle-repetitive DNA elements should be useful for fingerprint analysis in chickens, and in vertebrates in general.

Research note: a rapid method for the detection of the Rous-associated endogenous solitary long terminal repeat, ev15.

A rapid method has been developed for the detection of the solitary long terminal repeat ev15, a member of the avian leukosis virus (ALV) family of endogenous viral elements (ev genes) in chickens. Detection is accomplished by a polymerase chain reaction (PCR) assay that can be performed on purified genomic DNA samples or crude preparations of partially purified whole blood lysates. The test discriminates unambiguously between birds that are homozygous ev15-, homozygous ev15+, or heterozygous ev15-/ev15+. The incorporation of a modified touchdown amplification profile significantly improved the specificity of the PCR assay. Small-scale screening of birds from a variety of chicken breeds has revealed that ev15 is present in populations of both egg-strain birds and broilers.

Research note: avian leukosis retroviral genes are not detected in geese.

Genomic DNA from four strains of geese was analyzed for the presence of endogenous viral elements using a probe that can detect over 20 Rous-associated endogenous viral genes (ev genes) in chickens, as well as a probe and protocol that detects endogenous avian viruses (EAV). Southern blot analysis of genomic DNA did not reveal any ev genes in DNA of 15 geese from Chinese, Synthetic, or two Embden goose strains. Even under low stringency conditions, using a probe that covered most of the polymerase (pol) gene of the Rous-associated virus (RAV) and that revealed EAV elements in a chicken without ev genes, no viral loci were evident in goose DNA.