Blanca Lupiani

Rescue of a pathogenic Marek's disease virus with overlapping cosmid DNAs: Use of a pp38 mutant to validate the technology for the study of gene function

Mareks disease virus (MDV) genetics has lagged behind that of other herpesviruses because of the lack of tools for the introduction of site-specific mutations into the genome of highly cell-associated oncogenic strains. Overlapping cosmid clones have been successfully used for the introduction of mutations in other highly cell-associated herpesviruses. Here we describe the development of overlapping cosmid DNA clones from a very virulent oncogenic strain of MDV. Transfection of these cosmid clones into MDV-susceptible cells resulted in the generation of a recombinant MDV (rMd5) with biological properties similar to the parental strain. To demonstrate the applicability of this technology for elucidation of gene function of MDV, we have generated a mutant virus lacking an MDV unique phosphoprotein, pp38, which has previously been associated with the maintenance of transformation in MDV-induced tumor cell lines. Inoculation of Mareks disease-susceptible birds with the pp38 deletion mutant virus (rMd5Δpp38) revealed that pp38 is involved in early cytolytic infection in lymphocytes but not in the induction of tumors. This powerful technology will speed the characterization of MDV gene function, leading to a better understanding of the molecular mechanisms of MDV pathogenesis. In addition, because Mareks disease is a major oncogenic system, the knowledge obtained from these studies may shed light on the oncogenic mechanisms of other herpesviruses.

Identification of differentially expressed miRNAs in chicken lung and trachea with avian influenza virus infection by a deep sequencing approach

BackgroundMicroRNAs (miRNAs) play critical roles in a wide spectrum of biological processes and have been shown to be important effectors in the intricate host-pathogen interaction networks. Avian influenza virus (AIV) not only causes significant economic losses in poultry production, but also is of great concern to human health. The objective of this study was to identify miRNAs associated with AIV infections in chickens.ResultsTotal RNAs were isolated from lung and trachea of low pathogenic H5N3 infected and non-infected SPF chickens at 4 days post-infection. A total of 278,398 and 340,726 reads were obtained from lung and trachea, respectively. And 377 miRNAs were detected in lungs and 149 in tracheae from a total of 474 distinct chicken miRNAs available at the miRBase, respectively. Seventy-three and thirty-six miRNAs were differentially expressed between infected and non-infected chickens in lungs and tracheae, respectively. There were more miRNAs highly expressed in non-infected tissues than in infected tissues. Interestingly, some of these differentially expressed miRNAs, including miR-146, have been previously reported to be associated with immune-related signal pathways in mammals.ConclusionTo our knowledge, this is the first study on miRNA gene expression in AIV infected chickens using a deep sequencing approach. During AIV infection, many host miRNAs were differentially regulated, supporting the hypothesis that certain miRNAs might be essential in the host-pathogen interactions. Elucidation of the mechanism of these miRNAs on the regulation of host-AIV interaction will lead to the development of new control strategies to prevent or treat AIV infections in poultry.

Integrated analysis of microRNA expression and mRNA transcriptome in lungs of avian influenza virus infected broilers

BackgroundAvian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. Our previous study suggested microRNAs (miRNAs) play significant roles in the regulation of host response to AIV infection in layer chickens. The objective of this study was to test the hypothesis if genetic background play essential role in the miRNA regulation of AIV infection in chickens and if miRNAs that were differentially expressed in layer with AIV infection would be modulated the same way in broiler chickens. Furthermore, by integrating with parallel mRNA expression profiling, potential molecular mechanisms of host response to AIV infection can be further exploited.ResultsTotal RNA isolated from the lungs of non-infected and low pathogenic H5N3 infected broilers at four days post-infection were used for both miRNA deep sequencing and mRNA microarray analyses. A total of 2.6 M and 3.3 M filtered high quality reads were obtained from infected and non-infected chickens by Solexa GA-I Sequencer, respectively. A total of 271 miRNAs in miRBase 16.0 were identified and one potential novel miRNA was discovered. There were 121 miRNAs differentially expressed at the 5% false discovery rate by Fisher’s exact test. More miRNAs were highly expressed in infected lungs (108) than in non-infected lungs (13), which was opposite to the findings in layer chickens. This result suggested that a different regulatory mechanism of host response to AIV infection mediated by miRNAs might exist in broiler chickens. Analysis using the chicken 44 K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection.ConclusionsA comprehensive analysis combining both miRNA and targeted mRNA gene expression suggests that gga-miR-34a, 122–1, 122–2, 146a, 155, 206, 1719, 1594, 1599 and 451, and MX1, IL-8, IRF-7, TNFRS19 are strong candidate miRNAs or genes involved in regulating the host response to AIV infection in the lungs of broiler chickens. Further miRNA or gene specific knock-down assay is warranted to elucidate underlying mechanism of AIV infection regulation in the chicken.

Recombinant Marek's disease virus (MDV) lacking the Meq oncogene confers protection against challenge with a very virulent plus strain of MDV

Mareks disease virus (MDV) encodes a basic leucine-zipper protein, Meq, that shares homology with the Jun/Fos family of transcriptional factors. Conclusive evidence that Meq is an oncogene of MDV came from recent studies of a Meq-null virus, rMd5 Delta Meq. This virus replicated well in vitro, but was non-oncogenic in vivo. Further characterization of this virus in vivo indicated that the meq gene is dispensable for cytolytic infection since it replicated well in the lymphoid organs and feather follicular epithelium. Since rMd5 Delta Meq virus was apathogenic for chickens, we set out to investigate whether this virus could be a good candidate vaccine. Vaccine efficacy experiments conducted in Avian Disease and Oncology Laboratory (ADOL) 15I(5)x 7(1) chickens vaccinated with rMd5 Delta Meq virus or an ADOL preparation of CVI988/Rispens indicated that the Meq-null virus provided protection superior to CVI988/Rispens, the most efficacious vaccine presently available, following challenge with a very virulent (rMd5) and a very virulent plus (648A) MDV strains.

Detection and Characterization of a Distinct Bornavirus Lineage from Healthy Canada Geese (Branta canadensis)

ABSTRACT Avian bornaviruses (ABV), identified in 2008, infect captive parrots and macaws worldwide. The natural reservoirs of these viruses are unknown. Reverse transcription-PCR (RT-PCR) was used to screen oropharyngeal/cloacal swab and brain samples from wild Canada geese (Branta canadensis) for ABV. Approximately 2.9% of swab samples were positive for bornavirus sequences. Fifty-two percent of brain samples from 2 urban flocks also tested positive, and brain isolates were cultured in duck embryo fibroblasts. Phylogenetic analyses placed goose isolates in an independent cluster, and more notably, important regulatory sequences present in Borna disease virus but lacking in psittacine ABVs were present in goose isolates.

A multiplexed immunoassay for detection of antibodies against avian influenza virus.

Avian influenza (AI) is a highly contagious disease in poultry and outbreaks can have dramatic economic and health implications. For effective disease surveillance, rapid and sensitive assays are needed to detect antibodies against AI virus (AIV) proteins. In this study, we report the development of a multiplexed fluorescence microsphere immunoassay (FMIA) for detection of antibodies against AIV proteins in poultry. Recombinant nucleoprotein (NP), matrix protein (M1), and non-structural protein 1 (NS1) were expressed using a baculovirus expression system, purified and covalently coupled to fluorescent xMAP microspheres. Using these reagents, a triplex bead assay was developed for the Luminex platform. The assay displayed minimal cross reactivity when screened against a panel of reference sera raised against common avian viruses. For detection of anti-NP antibodies, the sensitivity and specificity of the assay were comparable to a commercially available ELISA. The assay was also employed to investigate the early kinetics of antibody response in chickens infected with AIV. Our results suggest that NP should be the protein of choice when detecting AI infections in commercial chickens, as the immune response was higher and persisted longer than that of M1 and NS1 proteins. This report provides a framework from which a more robust assay could be developed to profile exposure to many AIV subtypes in a single test.

New Syndrome of Mixed Bacterial and Viral Etiology in Cultured Turbot Scophthalmus maximus

Abstract A new disease occurred from August to October 1987 in cultured turbot in northwestern Spain. During the outbreak, there was continuous but low mortality, with a total cumulative loss of 4% of the affected population. Diseased turbot did not display unusual swimming behavior; the external signs of the disease were abdominal distension and hemorrhagic foci in the anus and base of the fins. Internal examination of affected fish revealed a swollen stomach and intestine, which were filled with a mucous liquid. The internal wall of the peritoneal cavity was hemorrhagic, and there was an accumulation of reddish fluid in the cavity. Bacteriological analysis revealed a sucrose-negative Vibrio in the kidney, liver, and spleen. This bacteriums biochemical and serological characteristics resembled those of V. splendidus. Samples of internal organs were processed for virological analysis and inoculated on chinook salmon embryo (CHSE-214) cells, which showed a cytopathic effect after 14 d at 15°C. The viral a...

Expansion of a Unique Region in the Marek's Disease Virus Genome Occurs Concomitantly with Attenuation but Is Not Sufficient To Cause Attenuation

ABSTRACT Pathogenic Mareks disease viruses (MDVs) have two head-to-tail copies of a 132-bp repeat. As MDV is serially passaged in cell culture, the virus becomes attenuated and the number of copies of the 132-bp repeat increases from 2 to often more than 20 copies. To determine the role of the repeats in attenuation, we used five overlapping cosmid clones that spanned the MDV genome to reconstitute infectious virus (rMd5). By mutating the appropriate cosmids, we generated clones of infectious MDVs that contained zero copies of the 132-bp repeats, rMd5(Δ132); nine copies of the 132-bp repeats, rMd5(9-132); and nine copies of the 132-bp repeats inserted in the reverse orientation, rMd5(rev9-132). After two passages in cell culture, wild-type Md5, rMd5, and rMd5(Δ132) were stable. However, rMd5(9-132) and rMd5(rev9-132) contained a population of viruses that contained from 3 to over 20 copies of the repeats. A major 1.8-kb mRNA, containing two copies of the 132-bp repeat, was present in wild-type Md5 and rMd5 but was not present in rMd5(Δ132), rMd5(9-132), rMd5(rev9-132), or an attenuated MDV. Instead, the RNAs transcribed from the 132-bp repeat region in rMd5(9-132) and rMd5(rev9-132) closely resembled the pattern of RNAs transcribed in attenuated MDVs. When inoculated into susceptible day-old chicks, all viruses produced various lesions. Thus, expansion of the number of copies of 132-bp repeats, which accompanies attenuation, is not sufficient in itself to attenuate pathogenic MDVs.

Multiyear surveillance for avian influenza virus in waterfowl from wintering grounds, Texas coast, USA.

This surveillance can help in assessments of the prevalence of wild animal-to-human transmission.

Diagnosis of Myeloid Leukosis Induced by a Recombinant Avian Leukosis Virus in Commercial White Leghorn Egg Laying Flocks

SUMMARY. Commercial white leghorn egg layer flocks being used to produce fertile eggs for human vaccine production exhibited dramatically low peaks in egg production, two to four times higher than normal weekly mortality, and high numbers of cull, nonlaying birds after the onset of sexual maturity. These lower production characteristics could not be associated with management-related problems. Gross lesions of cull and fresh dead birds necropsied showed approximately 60% lacked ovarian activity and had lesions of a bacterial bursitis or synovitis, whereas the other 40% had tumors of the viscera but not of the bursa of Fabricius. Histologic examination of tumor-containing tissues showed lesions typical of myelocytomatosis. The diagnosis of myeloid leukosis was confirmed by the isolation of a recombinant avian leukosis virus (ALV) containing the LTR of subgroup J and the envelope of subgroup B ALV. A positive polymerase chain reaction with primers specific for the 3′ untranslated region LTR confirmed the presence of LTR of ALV-J. The source of infection with this recombinant ALV was not determined; however, it is likely that commingling of the day-old egg-type chicks with ALV-J–infected meat-type chicks in a common hatchery had contributed to this outbreak.