Shirin Munir

Pathogenic role of SEF14, SEF17, and SEF21 fimbriae in Salmonella enterica serovar Enteritidis infection of chickens.

ABSTRACT Very little is known about the contribution of surface appendages of Salmonella enterica serovar Enteritidis to pathogenesis in chickens. This study was designed to clarify the role of SEF14, SEF17, and SEF21 fimbriae in serovar Enteritidis pathogenesis. Stable, single, defined sefA (SEF14), agfA (SEF17), andfimA (SEF21) insertionally inactivated fimbrial gene mutants of serovar Enteritidis were constructed. All mutant strains invaded Caco-2 and HT-29 enterocytes at levels similar to that of the wild type. Both mutant and wild-type strains were ingested equally well by chicken macrophage cell lines HD11 and MQ-NCSU. There were no significant differences in the abilities of these strains to colonize chicken ceca. The SEF14− strain was isolated in lower numbers from the livers of infected chickens and was cleared from the spleens faster than other strains. No significant differences in fecal shedding of these strains were observed.

Sequence analysis of the nucleocapsid and phosphoprotein genes of avian pneumoviruses circulating in the US

Avian pneumovirus (APV) has recently been described as the cause of a new respiratory syndrome in turkey flocks in the United States. We here describe the complete sequence of the nucleocapsid (N) and phosphoprotein (P) genes of this emerging APV (APV/US). Our results show 59 and 61% nucleotide sequence identity of the APV/US N gene with N genes of previously described European APV subgroups A and B, respectively. The P gene of APV/US showed only 53% nucleotide sequence identity with the ortholog from APV subgroup A. Phylogenetic analyses of both N and P genes clearly demonstrate that the APV/US lineage is evolutionarily related but distinct from European APVs. Moreover, sequence analysis of the N and P genes from two laboratory adapted isolates of APV/US (APV/MN-1a and APV/MN-1b) and from ten clinical samples from APV-infected turkeys suggests only modest level of amino acid divergence in the N (0-0.3%) and P (0-1.4%) proteins. Taken together, the results of this study indicate support that APV/US represents a new subgroup (subgroup C) of APV and show that there is limited heterogeneity in the N and P genes of APV/US isolates.

Identification and analysis of safener-inducible expressed sequence tags in Populus using a cDNA microarray

Safeners are the chemicals used to protect plants from detrimental effects of herbicides, but their mode of action at the molecular level is not well understood. As an initial step towards understanding the molecular mechanism of safener action in trees, homologous genes in hybrid poplar (Populus nigra × Populus maximowiczii) that were induced by a safener were identified. We here describe the identification of differentially expressed genes in Populus that are induced by Concep-III, a herbicide safener. Expressed sequence tags (ESTs) enriched for transcriptionally induced genes were isolated by suppressive subtractive hybridization (SSH). The SSH library cDNA inserts were used to construct a cDNA microarray for high-throughput validation of the up-regulated expression of safener-induced genes. Single-pass and partial sequences of 1,344 safener-induced ESTs were assembled into 418 singletons and 328 clusters, but the putative functions of almost 53% of the ESTs are not known. Genes encoding proteins involved in all three different phases of safener action, viz., oxidation, conjugation, and sequestration, were found in the SSH library. Almost 75% of genes that showed greater than 2-fold expression upon safener treatment were redundant in the SSH library. The expression pattern for selected genes was validated by reverse transcription–polymerase chain reaction. A few safener-induced genes that were not previously reported to be induced by safeners, but which may have a role in herbicide metabolism, were identified. The newly identified genes could have potential for application in genetic engineering of plants for herbicide detoxification and tolerance.

Transcriptional analysis of avian embryonic tissues following infection with avian infectious bronchitis virus

Abstract Avian infectious bronchitis virus (IBV) infection is one of the major viral respiratory diseases of chickens. Better understanding of the molecular basis of viral pathogenesis should contribute significantly towards the development of improved prophylactic, therapeutic and diagnostic reagents to control infections. In the present investigation, transcriptional profiles were analyzed by using RNA recovered from the lung tissue of IBV infected 18-day-old chicken embryos at 6, 24, 48 and 72h post IBV infection. This microarray analysis was completed using avian cDNA arrays comprised of fragments of 1191 unique chicken and turkey gene transcripts. These arrays were generated from normalized cDNA subtraction libraries that were derived from avian pneumovirus (APV) infected chicken embryo fibroblast (CEF) cultures and tissues obtained from APV infected turkeys subtracted with their respective uninfected cultures and tissues. Of the 1191 unique genes represented on the array, the expression of a total of 327 genes (27% of total) were altered by two-fold or more from 6 through 72h post-infection. A comparative analysis of IBV regulated genes with genes previously reported to change in expression following infection with other avian respiratory viruses revealed both conserved and unique changes. Real-time qRT-PCR was used to confirm the regulated expression of genes related to several functional classes including kinases, interferon induced genes, chemokines and adhesion molecules, vesicular trafficking and fusion protein genes, extracellular matrix protein genes, cell cycle, metabolism, cell physiology and development, translation, RNA binding, lysosomal, protein degradation and ubiquitination related genes. Microarray analysis served as an efficient tool in facilitating a comparative analysis of avian respiratory viral infections and provided insight into host transcriptional changes that were conserved as well as those which were unique to individual pathogens.

Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in virus infected cells

High throughput detection of differential expression of genes is an efficient means of identifying genes and pathways that may play a role in biological systems under certain experimental conditions. There exist a variety of approaches that could be used to identify groups of genes that change in expression in response to a particular stimulus or environment. We here describe the application of suppression subtractive hybridization (SSH) coupled with cDNA microarray analysis for isolation and identification of chicken transcripts that change in expression on infection of host cells with a paramyxovirus. SSH was used for initial isolation of differentially expressed transcripts, a large-scale validation of which was accomplished by microarray analysis. The data reveals a large group of regulated genes constituting many biochemical pathways that could serve as targets for future investigations to explore their role in paramyxovirus pathogenesis. The detailed methods described herein could be useful and adaptable to any biological system for studying changes in gene expression.

Detection of porcine teschovirus and enterovirus type II by reverse transcription-polymerase chain reaction.

Porcine enteroviruses (PEVs) have been recognized as the causative agents of various clinical manifestations such as fertility disorders, neurological defects, and dermal lesions in pigs. Currently, the diagnosis of PEV infection is carried out by virus isolation, which although useful, is labor- and time-intensive. The present investigation describes the development of a reverse transcription–polymerase chain reaction (RT-PCR) assay for the rapid and sensitive detection of PEVs of cytopathic effect groups I (now known as porcine teschoviruses [PTVs]) and II. The assay described not only detects the PTVs and CPE group II of PEVs but also allows them to be differentiated on the basis of the size of the amplification product, using the same set of oligonucleotide primers. The availability of specific and sensitive molecular diagnostic tools such as the RT-PCR assay described herein should facilitate efficient diagnosis of PTV and CPE group II infections in pigs.

Regulation of Host Cell Transcriptional Physiology by the Avian Pneumovirus Provides Key Insights into Host-Pathogen Interactions

ABSTRACT Infection with a viral pathogen triggers several pathways in the host cell that are crucial to eliminating infection, as well as those that are used by the virus to enhance its replication and virulence. We have here used suppression subtractive hybridization and cDNA microarray analyses to characterize the host transcriptional response in an avian pneumovirus model of infection. The results of our investigations reveal a dynamic host response that includes the regulation of genes with roles in a vast array of cellular functions as well as those that have not been described previously. The results show a considerable upregulation in transcripts representing the interferon-activated family of genes, predicted to play a role in virus replication arrest. The analysis also identified transcripts for proinflammatory leukocyte chemoattractants, adhesion molecules, and complement that were upregulated and may account for the inflammatory pathology that is the hallmark of viral respiratory infection. Interestingly, alterations in the transcription of several genes in the ubiquitin and endosomal protein trafficking pathways were observed, suggesting a role for these pathways in virus maturation and budding. Taken together, the results of our investigations provide key insights into individual genes and pathways that constitute the host cells response to avian pneumovirus infection, and they have enabled the development of resources and a model of host-pathogen interaction for an important avian respiratory tract pathogen.

Detection of Antibodies to U.S. Isolates of Avian Pneumovirus by a Recombinant Nucleocapsid Protein-Based Sandwich Enzyme-Linked Immunosorbent Assay

ABSTRACT The nucleocapsid (N) protein of subgroup C (United States-specific) avian pneumovirus (APV/US) was expressed inEscherichia coli, and antibodies to the recombinant N protein were shown to specifically recognize the ≈47-kDa N protein of APV/US by Western immunoblot analysis. The recombinant APV/US N protein was used in a sandwich-capture enzyme-linked immunosorbent assay (ELISA), and the resulting assay was found to be more sensitive and specific than the routine indirect ELISA for the detection of APV/US antibodies in turkey sera.

PCR-Based Detection of an Emerging Avian Pneumovirus in US Turkey Flocks

Avian pneumovirus (APV) or turkey rhinotracheitis virus (TRTV) is an important respiratory pathogen of domesticated poultry in many countries in Europe, Africa, and Asia. Until recently, the United States was considered free of APV. In late 1996, an atypical upper respiratory tract infection appeared in turkey flocks in Colorado and shortly thereafter in turkey flocks in Minnesota. An avian pneumovirus (APV-US) that was serologically distinct from the previously described TRTV was isolated as the primary cause of the new syndrome. The nucleotide sequence of a fragment of the APV-US fusion gene was determined and used to develop a polymerase chain reaction–based assay that specifically detects APV-US viral nucleic acid sequences in RNA extracts of tracheal swabs and turbinate homogenates. The assay is highly sensitive in that it can detect <0.01 TCID50 of APV. The availability of this assay enables the rapid and accurate determination of APV-US in infected poultry flocks.

Rapid Expression of Mycobacterium avium subsp. paratuberculosis Recombinant Proteins for Antigen Discovery

ABSTRACT Mycobacterium avium subsp. paratuberculosis is the causative agent of Johnes disease, a chronic granulomatous enteritis of ruminants and other species. Detection of infection in animals is hampered by the lack of sensitive and specific diagnostic assays. We describe here an approach that utilizes translationally active PCR fragments for the rapid in vitro transcription and translation of recombinant proteins for antigen discovery in M. avium subsp. paratuberculosis. The investigations showed that the MAP1272c protein selectively reacts with sera from Johnes disease-positive cattle and represents an antigen of potential utility in M. avium subsp. paratuberculosis immunodiagnostics.