Background

The role of community pharmacists in disease state management has been mooted for some years. Despite a number of trials of disease state management services, there is scant literature into the engagement of, and with, pharmacists in such trials. This paper reports pharmacists’ feedback as providers of a Pharmacy Asthma Management Service (PAMS), a trial coordinated across four academic research centres in Australia in 2009. We also propose recommendations for optimal involvement of pharmacists in academic research.

Methods

Feedback about the pharmacists’ experiences was sought via their participation in either a focus group or telephone interview (for those unable to attend their scheduled focus group) at one of three time points. A semi-structured interview guide focused discussion on the pharmacists’ training to provide the asthma service, their interactions with health professionals and patients as per the service protocol, and the future for this type of service. Focus groups were facilitated by two researchers, and the individual interviews were shared between three researchers, with data transcribed verbatim and analysed manually.

Results

Of 93 pharmacists who provided the PAMS, 25 were involved in a focus group and seven via telephone interview. All pharmacists approached agreed to provide feedback. In general, the pharmacists engaged with both the service and research components, and embraced their roles as innovators in the trial of a new service. Some experienced challenges in the recruitment of patients into the service and the amount of research-related documentation, and collaborative patient-centred relationships with GPs require further attention. Specific service components, such as the spirometry, were well received by the pharmacists and their patients. Professional rewards included satisfaction from their enhanced practice, and pharmacists largely envisaged a future for the service.

Conclusions

The PAMS provided pharmacists an opportunity to become involved in an innovative service delivery model, supported by the researchers, yet trained and empowered to implement the clinical service throughout the trial period and beyond. The balance between support and independence appeared crucial in the pharmacists’ engagement with the trial. Their feedback was overwhelmingly positive, while useful suggestions were identified for future academic trials.

Lymphoid oncogenesis is a life threatening complication associated with a number of persistent viral infections (e.g. EBV and HTLV-1 in humans). With many of these infections it is difficult to study their natural history and the dynamics of tumor formation. Marek's Disease Virus (MDV) is a prevalent α-herpesvirus of poultry, inducing CD4+ TCRαβ+ T cell tumors in susceptible hosts. The high penetrance and temporal predictability of tumor induction raises issues related to the clonal structure of these lymphomas. Similarly, the clonality of responding CD8 T cells that infiltrate the tumor sites is unknown. Using TCRβ repertoire analysis tools, we demonstrated that MDV driven CD4+ T cell tumors were dominated by one to three large clones within an oligoclonal framework of smaller clones of CD4+ T cells. Individual birds had multiple tumor sites, some the result of metastasis (i.e. shared dominant clones) and others derived from distinct clones of transformed cells. The smaller oligoclonal CD4+ cells may represent an anti-tumor response, although on one occasion a low frequency clone was transformed and expanded after culture. Metastatic tumor clones were detected in the blood early during infection and dominated the circulating T cell repertoire, leading to MDV associated immune suppression. We also demonstrated that the tumor-infiltrating CD8+ T cell response was dominated by large oligoclonal expansions containing both “public” and “private” CDR3 sequences. The frequency of CD8+ T cell CDR3 sequences suggests initial stimulation during the early phases of infection. Collectively, our results indicate that MDV driven tumors are dominated by a highly restricted number of CD4+ clones. Moreover, the responding CD8+ T cell infiltrate is oligoclonal indicating recognition of a limited number of MDV antigens. These studies improve our understanding of the biology of MDV, an important poultry pathogen and a natural infection model of virus-induced tumor formation.

Author Summary

Many viral infections target the immune system, making use of the long lived, highly proliferative lymphocytes to propagate and survive within the host. This characteristic has led to an association between some viruses such as Epstein Barr Virus (EBV), Human T cell Lymphotrophic Virus-1 (HTLV-1) and Mareks Disease Virus (MDV) and lymphoid tumors. We employed methods for identifying the T cell receptor repertoire as a molecular bar-code to study the biology of MDV-induced tumors and the anti-tumor response. Each individual contained a small number of large (high frequency) tumor clones alongside some smaller (lower frequency) clones in the CD4+ T cell population. The tumor infiltrating CD8+ T cell response was highly focused with a small number of large clones, with one representing a public CDR3 sequence. This data is consistent with the recognition of a small number of dominant antigens and understanding the relationship between these and protective immunity is important to improve development of new vaccination strategies. Collectively, our results provide insights into the clonal structure of MDV driven tumors and in the responding CD8+ T cell compartment. These studies advance our understanding of MDV biology, an important poultry disease and a natural infection model of virus-induced tumor formation.

Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.

Author Summary

MicroRNAs (miRNAs), encoded in the genomes of a number of organisms including several viruses, belong to a class of small RNA molecules that can function as key regulators of gene expression influencing various biological processes and diseases including cancer. Among all the miRNAs, miR-155 has been well documented for its direct role of oncogenesis in a number of species including chickens. Remarkably, miR-K12-11 and miR-M4, the miRNAs encoded by the oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) and Marek's disease virus (MDV) respectively, have been shown to be functional orthologs of miR-155. There are no animal models of KSHV-induced tumors to examine the oncogenic potential of miR-K12-11. However, using recombinant mutant viruses in excellent models of MDV-induced lymphomas in their natural chicken hosts, we demonstrate that miR-M4 is critical for the induction of tumors. This is the first study that clearly demonstrates a direct role for a single miRNA in inducing cancer in an in vivo animal model. The ability of gga-miR-155 to rescue the oncogenic potential of miR-M4-deleted virus demonstrated the conservation of oncogenic functions the two miRNAs. Moreover, we show that virus attenuated by deleting the miRNAs can function as vaccines against virulent virus infection, enabling the prospects of generating novel molecularly-defined vaccines.

Bacterial artificial chromosome (BAC) vectors containing the full-length genomes of several herpesviruses have been used widely as tools to enable functional studies of viral genes. Marek's disease viruses (MDVs) are highly oncogenic alphaherpesviruses that induce rapid-onset T-cell lymphomas in chickens. Oncogenic strains of MDV reconstituted from BAC clones have been used to examine the role of viral genes in inducing tumours. Past studies have demonstrated continuous increase in virulence of MDV strains. We have previously reported on the UK isolate C12/130 that showed increased virulence features including lymphoid organ atrophy and enhanced tropism for the central nervous system. Here we report the construction of the BAC clones (pC12/130) of this strain. Chickens were infected with viruses reconstituted from the pC12/130 clones along with the wild-type virus for the comparison of the pathogenic properties. Our studies show that BAC-derived viruses induced disease similar to the wild-type virus, though there were differences in the levels of pathogenicity between individual viruses. Generation of BAC clones that differ in the potential to induce cytolytic disease provide the opportunity to identify the molecular determinants of increased virulence by direct sequence analysis as well as by using reverse genetics approaches on the infectious BAC clones.

Objectives

To compare longitudinal data with previous cross-sectional data regarding Australian undergraduate pharmacy students' approaches to learning, and explore the differences in approaches to learning between undergraduate and postgraduate cohorts.

Methods

Longitudinal, repeated measures design using a validated self-report survey instrument were used to gather data.

Results

Undergraduate students' preferences for meaning directed, undirected, and reproduction-directed approaches to learning displayed the same pattern across the 2 studies; however, application-directed scores increased significantly in the second half of the undergraduate degree program. Commencing postgraduate students' approaches to learning were similar to finishing undergraduate students, and this group was significantly more oriented towards meaning-directed learning compared to undergraduate students.

Conclusions

Pharmacy students' maturation in approach to their learning was evident and this bodes well for pharmacists' engaging in life-long learning and capacity to work in increasingly complex health settings.

Background

Despite national disease management plans, optimal asthma management remains a challenge in Australia. Community pharmacists are ideally placed to implement new strategies that aim to ensure asthma care meets current standards of best practice. The impact of the Pharmacy Asthma Care Program (PACP) on asthma control was assessed using a multi‐site randomised intervention versus control repeated measures study design.

Methods

Fifty Australian pharmacies were randomised into two groups: intervention pharmacies implemented the PACP (an ongoing cycle of assessment, goal setting, monitoring and review) to 191 patients over 6 months, while control pharmacies gave their usual care to 205 control patients. Both groups administered questionnaires and conducted spirometric testing at baseline and 6 months later. The main outcome measure was asthma severity/control status.

Results

186 of 205 control patients (91%) and 165 of 191 intervention patients (86%) completed the study. The intervention resulted in improved asthma control: patients receiving the intervention were 2.7 times more likely to improve from “severe” to “not severe” than control patients (OR 2.68, 95% CI 1.64 to 4.37; p<0.001). The intervention also resulted in improved adherence to preventer medication (OR 1.89, 95% CI 1.08 to 3.30; p = 0.03), decreased mean daily dose of reliever medication (difference −149.11 μg, 95% CI −283.87 to −14.36; p = 0.03), a shift in medication profile from reliever only to a combination of preventer, reliever with or without long‐acting β agonist (OR 3.80, 95% CI 1.40 to 10.32; p = 0.01) and improved scores on risk of non‐adherence (difference −0.44, 95% CI −0.69 to −0.18; p = 0.04), quality of life (difference −0.23, 95% CI −0.46 to 0.00; p = 0.05), asthma knowledge (difference 1.18, 95% CI 0.73 to 1.63; p<0.01) and perceived control of asthma questionnaires (difference −1.39, 95% CI −2.44 to −0.35; p<0.01). No significant change in spirometric measures occurred in either group.

Conclusions

A pharmacist‐delivered asthma care programme based on national guidelines improves asthma control. The sustainability and implementation of the programme within the healthcare system remains to be investigated.

Background

Micro(mi)RNAs are a class of small non-coding RNAs that play critical roles in the induction of various cancers, including lymphomas induced by oncogenic viruses. While some of the miRNAs are oncogenic, miRNAs such as miR-26a are consistently downregulated in a number of cancers, demonstrating their potential tumor suppressor functions. Global miRNA expression profiles of a number of virus-transformed avian lymphoma cell lines have shown downregulation of gga-miR-26a expression, irrespective of molecular mechanisms of transformation or the viral aetiology. The neoplastic transformation of lymphocytes by many viruses accompanies high levels of proliferative responses, mostly mediated through cytokines such as IL-2. Chicken IL-2 can modulate T-cell proliferation and cytotoxicity in vitro and in vivo and dysregulation of IL-2 expression is observed in diseases such as leukaemia.

Results

The expression levels of gga-miR-26a in chicken lymphoma cells transformed by 3 distinct avian oncogenic viruses, viz Marek's disease virus (MDV), avian leukosis virus (ALV) and Reticuloendotheliosis virus (REV) were consistently downregulated compared to the levels in the normal lymphocytes. This downregulation of miR-26a regardless of the viral etiology and molecular mechanisms of transformation was consistent with the tumor suppressor role of this miRNA. Notwithstanding this well-established role in cancer, we demonstrate the additional role of this miRNA in directly targeting chicken IL-2 through reporter and biochemical assays. The downregulation of miR-26a can relieve the suppressive effect of this miRNA on IL-2 expression.

Conclusions

We show that miR-26a is globally downregulated in a number of avian lymphoma cells irrespective of the mechanisms of transformation, reiterating the highly conserved tumor suppressor function of this miRNA. However, with the potential for directly targeting chicken IL-2, the downregulation of miR-26a in these tumor cells could relieve the inhibitory effect on IL-2 expression assisting in the proliferative features of the transformed lymphocyte lines.

Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus that induces fatal rapid-onset T-cell lymphomas in chickens, its natural host. The MDV-encoded nuclear oncoprotein Meq is essential for lymphomagenesis and acts as a regulator of transcription. Meq has structural features, including a basic domain adjacent to a leucine zipper motif (B-ZIP), that suggest it is related to the Jun/Fos family of transcription factors. Via the leucine zipper, Meq can form homodimers or heterodimerize with c-Jun. Meq/Meq homodimers are associated with transrepression, and Meq/Jun heterodimers can transactivate target genes carrying an AP-1-like binding site. In order to determine the role of the leucine zipper and of Meq dimerization in T lymphomagenesis, specific point mutations were engineered into the highly oncogenic RB-1B strain of MDV to produce virus completely lacking a functional Meq leucine zipper (RB-1B MeqBZIP/BZIP) or virus encoding Meq that cannot homodimerize but can still bind to c-Jun and an AP-1-like site on DNA (RB-1B MeqHom/Hom). Both of these mutant viruses were capable of replication in cultured chicken embryo fibroblasts. However both mutations resulted in a complete loss of oncogenicity, since no lymphomas were produced up to 90 days postinfection in experimentally infected chicks. We conclude that the leucine zipper is necessary for the oncogenic activity of Meq and/or the efficient establishment of long-term MDV latency in T cells. Moreover, it appears that the ability to form homodimers is an absolute requirement and the ability to bind c-Jun alone is insufficient for the T-cell lymphomagenesis associated with virulent MDV.

Herpesviruses account for 134 out of the 140 virus-encoded microRNAs (miRNAs) known today. Here we report the identification of 11 novel miRNAs encoded by herpesvirus of turkey (HVT), a virus used as a live vaccine in poultry against the highly oncogenic Marek's disease virus type 1. Ten of these miRNAs were clustered together within the repeat long region of the viral genome, demonstrating some degree of positional conservation with other mardiviruses. Close sequence and phylogenetic relationships of some miRNAs in this cluster indicate evolution by duplication. HVT miRNAs represent the first example of virus-encoded miRNAs that show evolution by duplication.

In this study, we have identified an internal ribosome entry site (IRES) from the highly infectious herpesvirus Marek's disease virus (MDV). The IRES was mapped to the intercistronic region (ICR) of a bicistronic mRNA that we cloned from the MDV-transformed CD4+ T-cell line MSB-1. The transcript is a member of a family of mRNAs expressed as immediate-early genes with two open reading frames (ORF). The first ORF encodes a 14-kDa polypeptide with two N-terminal splice variants, whereas the second ORF is contained entirely within a single exon and encodes a 12-kDa protein also known as RLORF9. We have shown that the ICR that separates the two ORFs functions as an IRES that controls the translation of RLORF9 when cap-dependent translation is inhibited. Deletion analysis revealed that there are two potential IRES elements within the ICR. Reverse genetic experiments with the oncogenic strain of MDV type 1 indicated that deletion of IRES-controlled RLORF9 does not significantly affect viral replication or MDV-induced mortality.

Objectives

To assess undergraduate and postgraduate pharmacy students' perceptions of plagiarism and academic honesty.

Methods

A questionnaire was administered to undergraduate and postgraduate pharmacy students to determine their levels of awareness of university policy concerning academic honesty; attitudes to plagiarism by rating the acceptability of a range of plagiarizing and cheating practices; and choice of appropriate penalties for a first and second occurrence. The choice of behaviors in response to a scenario about the preparation of a reading-based written assignment and the strategies that students would be prepared to use in order to submit the assignment on time were also assessed.

Results

Findings indicated widespread deficiencies in student knowledge of, and attitudes towards, plagiarism. Students did not perceive plagiarism as a serious issue and the use of inappropriate strategies for sourcing and acknowledging material was common.

Conclusions

The study highlights the importance of achieving a balance among the 3 dimensions of plagiarism management: prevention, detection and penalty.

Kaposi's sarcoma-associated herpesvirus-encoded microRNA (miRNA) MiR-K12-11 was recently shown to be a functional ortholog of miR-155, a miRNA that plays a major role in lymphoid malignancies and the modulation of immune responses. Here we show that miR-M4, encoded by the highly oncogenic Marek's disease virus of chickens, shares common targets with miR-155 and thus is also a functional ortholog of miR-155, the first one identified in an alphaherpesvirus. The observation that two distinct oncogenic herpesviruses associated with distinct types of lymphomas in different species encode functional miR-155 orthologs suggested the importance of this miRNA in regulatory pathways and the biology of lymphomagenesis.

Objectives

To identify best practices in global pharmacy education and curriculum design as the basis for decisions about major curriculum change in an existing 4-year bachelor of pharmacy curriculum.

Design

We investigated international best-practice standards, conducted semi-structured interviews with faculty members, and used standardized instruments to investigate student perceptions of the existing curriculum and how they approached their learning.

Assessment

Faculty recommendations included horizontal and vertical integration of curriculum content to replace the previous discipline-based approach; and a theme-based structure underpinned by a detailed statement of learning outcomes that describe the knowledge, skills, and attitudinal milestones to be achieved each year and by the time of graduation. The triangulation of student survey data highlighted issues that needed to be addressed at the individual course unit level, with a particular focus on feedback, assessment, and workload.

Conclusions

The results of the curriculum review provided clear guidance for decisions relating to major curriculum change. An ongoing program of staff development will address the wide range of learning and teaching issues identified by both staff members and students. The results of our investigation of students' approaches to learning will also be used to guide staff development workshops, focusing on strategies to promote “meaningful learning.”

Research over the last few years has demonstrated the increasing role of microRNAs (miRNAs) as major regulators of gene expression in diverse cellular processes and diseases. Several viruses, particularly herpesviruses, also use the miRNA pathway of gene regulation by encoding their own miRNAs. Marek's disease (MD) is a widespread lymphomatous neoplastic disease of poultry caused by the highly contagious Marek's disease virus type 1 (MDV-1). Recent studies using virus-infected chicken embryo fibroblasts have identified at least eight miRNAs that map to the RL/RS region of the MDV genome. Since MDV is a lymphotropic virus that induces T-cell lymphomas, analysis of the miRNA profile in T-cell lymphoma would be more relevant for examining their role in oncogenesis. We determined the viral and host miRNAs expressed in MSB-1, a lymphoblastoid cell line established from an MDV-induced lymphoma of the spleen. In this paper, we report the identification of 13 MDV-1-encoded miRNAs (12 by direct cloning and 1 by Northern blotting) from MSB-1 cells. These miRNAs, five of which are novel MDV-1 miRNAs, map to the Meq and latency-associated transcript regions of the MDV genome. Furthermore, we show that miRNAs encoded by MDV-1 and the coinfected MDV-2 accounted for >60% of the 5,099 sequences of the MSB-1 “miRNAome.” Several chicken miRNAs, some of which are known to be associated with cancer, were also cloned from MSB-1 cells. High levels of expression of MDV-1-encoded miRNAs and potentially oncogenic host miRNAs suggest that miRNAs may have major roles in MDV pathogenesis and neoplastic transformation.

Cloning of full length genomes of herpesviruses as bacterial artificial chromosomes (BAC) has greatly facilitated the manipulation of the genomes of several herpesviruses to identify the pathogenic determinants. We have previously reported the construction of the BAC clone (pRB-1B5) of the highly oncogenic Marek's disease virus (MDV) strain RB-1B, which has proven to be a valuable resource for elucidating several oncogenic determinants. Despite the retention of the BAC replicon within the genome, the reconstituted virus was able to induce tumours in susceptible chickens. Nevertheless, it was unclear whether the presence of the BAC influenced the full oncogenic potential of the reconstituted virus. To maximize the closeness of BAC-derived virus to the parental RB-1B strain, we modified the existing pRB-1B5 clone by restoring the Us2 and by introducing SV40-cre cassette within the loxP sites of the mini-F plasmid, to allow self-excision of the plasmid sequences in chicken cells. The reconstituted virus from the modified clone showed significant improvement in replication in vitro and in vivo. Excision of the BAC sequences also enhanced the pathogenicity to levels similar to that of the parental virus, as the cumulative incidence of Marek's disease in groups infected with the recombinant and the parental viruses showed no significant differences. Thus, we have been able to make significant improvements to the existing BAC clone of this highly oncogenic virus which would certainly increase its usefulness as a valuable tool for studies on identifying the oncogenic determinants of this major avian pathogen.

Objectives

To investigate how pharmacy students' approaches to learning change over the duration of a bachelor of pharmacy degree program.

Methods

Data were obtained from a cross-sectional, repeated measures design, using a validated self-report survey instrument. Areas examined included processing and regulation strategies, motivational preferences for learning, and the relationship between approaches to learning and academic performance.

Results

Pharmacy students were strongly vocationally oriented in their studies across all year groups. This approach had a significant relationship to academic performance. Overall, students indicated a preference for external regulation strategies. There was little evidence of maturation in approaches to learning as students progressed through the curriculum.

Conclusions

Students' preference for vocationally related strategies can be harnessed to increase both adoption of self-regulation behaviors and motivation for mastery of material. Comparison of our results with other studies indicates that approaches to learning may be influenced more by the learning environment than the discipline of study.

MicroRNAs (miRNAs) are increasingly being recognized as major regulators of gene expression in many organisms, including viruses. Among viruses, members of the family Herpesviridae account for the majority of the currently known virus-encoded miRNAs. The highly oncogenic Marek's disease virus type 1 (MDV-1), an avian herpesvirus, has recently been shown to encode eight miRNAs clustered in the MEQ and LAT regions of the viral genome. The genus Mardivirus, to which MDV-1 belongs, also includes the nononcogenic but antigenically related MDV-2. As MDV-1 and MDV-2 are evolutionarily very close, we sought to determine if MDV-2 also encodes miRNAs. For this, we cloned, sequenced, and analyzed a library of small RNAs from the lymphoblastoid cell line MSB-1, previously shown to be coinfected with both MDV-1 and MDV-2. Among the 5,099 small RNA sequences determined from the library, we identified 17 novel MDV-2-specific miRNAs. Out of these, 16 were clustered in a 4.2-kb long repeat region that encodes R-LORF2 to R-LORF5. The single miRNA outside the cluster was located in the short repeat region, within the C-terminal region of the ICP4 homolog. The expression of these miRNAs in MSB-1 cells and infected chicken embryo fibroblasts was further confirmed by Northern blotting analysis. The identification of miRNA clusters within the repeat regions of MDV-2 demonstrates conservation of the relative genomic positions of miRNA clusters in MDV-1 and MDV-2, despite the lack of sequence homology among the miRNAs of the two viruses. The identification of these novel miRNAs adds to the growing list of virus-encoded miRNAs.

Objectives

The development, implementation, and evaluation of an educational intervention to facilitate specialized asthma care provision by community pharmacists.

Design

Formative evaluation and a parallel group repeated measures design were used to test the effect of an educational intervention on pharmacist satisfaction and practice behavior as well as patient outcomes. The educational intervention was based on practitioner needs and principles of adult learning using flexible delivery formats.

Assessment

In the intervention area, 15 pharmacists were trained with the educational intervention, and they provided specialized asthma care to 52 patients over 6 months, while in the control area, 12 pharmacists provided “usual care” to 50 patients. The intervention pharmacists were highly satisfied with the education received and rated most aspects highly. Improvements in patient clinical, humanistic, and economic outcomes in the intervention area were obtained.

Conclusion

The positive results of the educational intervention demonstrate the effectiveness of an educational approach grounded in the theory that inducing behavioral changes in pharmacy practitioners results in improved patient outcomes.

Avian leukosis virus (ALV) subgroup J is thought to have emerged through a recombination event between an unknown exogenous ALV and the endogenous retrovirus elements designated EAV-HP. All EAV-HP elements identified to date in the chicken genome show large deletions, including that of the entire pol gene. Here we report the identification of four segregating chicken EAV-HP proviruses with complete pol genes, one of which shows exceptionally high sequence identity and a close phylogenetic relationship with ALV-J with respect to the env gene. Embryonic expression of EAV-HP env has been suggested as a factor associated with immunological tolerance induction in a proportion of ALV-J-infected meat-type chickens. In support of this, env gene transcripts expressed from two of the four newly identified EAV-HP proviruses were demonstrated in chicken embryos. However, when ALV-J-infected outbred meat-type chickens were assessed, the presence of intact EAV-HP proviruses failed to directly correlate with ALV-J tolerance. This association was further examined using F2 progeny of two inbred lines of layer chicken that differed in EAV-HP status and immunological responses to ALV-J. Immunological tolerance developed in a small proportion of F2 progeny birds, reflecting the expected phenotypic ratio for inheritance of a double-recessive genotype; however, the status of tolerance did not show any direct correlation with the presence of the intact EAV-HP sequence. Nevertheless, identification of an intact chicken EAV-HP locus showing a uniquely close relationship to the ALV-J prototype clone HPRS-103 in the env region provides the strongest evidence of its contribution to the emergence of ALV-J by recombination.