Transcription Mapping and Characterization of 284R and 121R Proteins Produced from Early Region 3 of Bovine Adenovirus Type 3

AbstractWe established the transcription map of early region (E) 3 of bovine adenovirus 3 (BAV-3) by Northern blot, S1 nuclease protection assays, cDNA sequencing, and RT-PCR analysis. Five major classes of mRNAs were identified, which shared the 3′ ends. Four classes of mRNAs transcribed from the E3 promoter also shared the 5′ end, while one major class of mRNA transcribed from the major late promoter contained a tripartite leader sequence at the 5′ end. These five transcripts have the potential to encode four proteins, namely 284R, 121R, 86R, and 82R. To identify the proteins, rabbit antiserum was prepared using a bacterial fusion protein encoding 284R or 121R protein. Serum against 284R immunoprecipitated protein of 26–32 kDa in in vitro translated and transcribed mRNA and three proteins of 48, 67, and 125 kDa from BAV-3-infected cells. Western blots and enzymatic digestions confirmed that the 284R protein is a glycoprotein, which contains only N-linked oligosaccharides, both high mannose (48 kDa) and complex types (67 kDa). Serum against 121R immunoprecipitated a protein of 14.5 kDa from in vitro translated and transcribed mRNA and BAV-3-infected cells. Although 121R protein shows limited sequence similarity to a 14.7-kDa protein of human adenovirus 5, the 284R protein appears to be unique to BAV-3. Since proteins encoded by the E3 region appear to influence adenovirus pathogenesis, the 284R protein may contribute to the unique pathogenic properties of BAV-3

Expression of Bovine Interleukin-1β in a Bovine Herpesvirus-1 Vector:In VitroAnalysis

AbstractIn order to evaluate whether bovine herpesvirus-1 (BHV-1) could be used as a live viral vector for the expression of cytokines, we constructed a recombinant BHV-1 expressing bovine interleukin-1β (boIL-1β). The boIL-1β coding sequence, corresponding to the cleaved mature product, was fused with the BHV-1 glycoprotein C (gC) signal peptide sequence; the resultant gC-boIL-1β fusion gene was recombined into the gC locus of the BHV-1 genome. Southern blot analysis confirmed the proper genomic configuration of the recombinant virus. Results from transcript analysis showed that boIL-1β was expressed in infected cells with kinetics similar to that of gC. Indirect immunofluorescence and immunoprecipitation assays showed that the recombinant protein was produced in both cell-associated and secreted forms. Western blot analysis detected a 19.3-kDa protein. Further analysis, using an IL-1β bioassay demonstrated that both the cellular and secreted forms of recombinant boIL-1β possessed biological activity. The expression of the boIL-1β protein did not affect thein vitrogrowth efficiency of the virus, which exhibited similar growth kinetics to that of a simple gC deletion mutant. The results from this study demonstrate that BHV-1 can be used to express a functional cytokine, thereby establishing the basis to further study recombinant BHV-1 expressing cytokines as an alternative means to attenuate the virus and also as a potentialin situcytokine delivery system to modulate immune responses against BHV-1 and other cattle pathogens

Reactivation of temperature-sensitive and non-temperature-sensitive infectious bovine rhinotracheitis vaccine virus with dexamethasone

Latent infections by a temperature-sensitive (ts) infectious bovine rhinotracheitis virus vaccine was produced as frequently as by non-ts vaccine virus. Thus, virus could be reactivated in seven of eight ts vaccinates and six of eight non-ts vaccinates after dexamethasone treatment. Virus excretion could be detectable for 1 to 8 days at a level of 2 X 106 to 3 X 108 plaque-forming units per ml of nasal secretions. The reactivated virus was shown to be the same as the original virus used for vaccination by its inability to grow at the restrictive temperature (39°C) as well as by its restriction endonuclease cleavage pattern

Pathogenesis and Immunogenicity of Bovine Adenovirus Type 3 in Cotton Rats (Sigmodon hispidus)

AbstractIntranasal inoculation of cotton rats (Sigmodon hispidus) with 108 PFU of bovine adenovirus type 3 (BAd3) resulted in limited virus replication in the lung and trachea. Histopathological changes in the lungs were characterized by necrosis and hyperplasia of bronchiolar epithelium, eosinophilic intranuclear inclusions, pneumocyte type II hyperplasia in the alveoli, and mild peribronchiolar and perivascular lymphocytic infiltration. Immunohistochemically, viral antigens were observed more frequently in bronchiolar epithelial cells than in alveolar cells in cotton rat lung sections stained using a rabbit anti-BAd3 serum. Bronchiolar epithelial changes, intranuclear inclusion bodies, type II pneumocyte proliferation, peribronchiolar infiltration, and immunohistological staining were maximum at Day 3 or Day 4 postinoculation, whereas perivascular infiltration was first observed at Day 8 postinoculation. In addition to the histological study of the pathogenesis of BAd3 infection, we monitored the BAd3-specific immune response in cotton rats. Anti-BAd3 IgG and virus neutralizing antibodies were detected in sera, whereas anti-BAd3 IgA antibodies were found in the sera, lung, and nasal washes. Our results suggest that the cotton rat can serve as a useful small-animal model for investigating the pathogenesis of BAd3 infection, as well as immune responses to BAd3 recombinant virus vaccines

Streptococcus pneumoniae early response genes to human lung epithelial cells

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus pneumoniae </it>infection starts from colonization of the host respiratory tract where interaction with host respiratory tract epithelial cells occurs. To investigate pneumococcal genes that are involved in the early stage of interaction with host epithelial cells, transcriptional responses of an encapsulated pathogenic pneumococcal strain TIGR4 upon exposure to human lung epithelial cells A549 for 0.5 h and 1 h time periods were investigated by using TIGR (JCVI) microarray technology. Gene expression changes were validated by quantitative real-time PCR (qRT-PCR) analysis.</p> <p>Findings</p> <p>We observed different transcriptional profiles at two incubation time periods in which most gene expressions were down-regulated at 0.5 h but up-regulated at 1 h. Many genes associated with ribonucleotide biosynthesis were down-regulated at both time points, whereas the genes associated with cell envelope, energy metabolism, transport and protein synthesis were mostly up-regulated at 1 h. Furthermore, these profiles were compared to the transcriptomes of a TIGR4-derived strain in response to human macrophages for the same time periods. We found one set of genes that exhibited similar expression changes upon exposure to both types of host cells, including cell envelope-associated <it>bgaA </it>(SP0648) and <it>nanA </it>(SP1693), and uncharacterized gene clusters such as SP1677–SP1680 and SP1688–SP1690.</p> <p>Conclusion</p> <p>These data indicate that at the early stage of interaction with host epithelial cells, a complex gene regulation and expression change occur in bacteria. Some of them might play an essential role during pathogen-host interactions and for the establishment of infection.</p

Stress significantly increases mortality following a secondary bacterial respiratory infection

A variety of mechanisms contribute to the viral-bacterial synergy which results in fatal secondary bacterial respiratory infections. Epidemiological investigations have implicated physical and psychological stressors as factors contributing to the incidence and severity of respiratory infections and psychological stress alters host responses to experimental viral respiratory infections. The effect of stress on secondary bacterial respiratory infections has not, however, been investigated. A natural model of secondary bacterial respiratory infection in naive calves was used to determine if weaning and maternal separation (WMS) significantly altered mortality when compared to calves pre-adapted (PA) to this psychological stressor. Following weaning, calves were challenged with Mannheimia haemolytica four days after a primary bovine herpesvirus-1 (BHV-1) respiratory infection. Mortality doubled in WMS calves when compared to calves pre-adapted to weaning for two weeks prior to the viral respiratory infection. Similar results were observed in two independent experiments and fatal viral-bacterial synergy did not extend beyond the time of viral shedding. Virus shedding did not differ significantly between treatment groups but innate immune responses during viral infection, including IFN-γ secretion, the acute-phase inflammatory response, CD14 expression, and LPS-induced TNFα production, were significantly greater in WMS versus PA calves. These observations demonstrate that weaning and maternal separation at the time of a primary BHV-1 respiratory infection increased innate immune responses that correlated significantly with mortality following a secondary bacterial respiratory infection

Construction and Characterization of E3-Deleted Bovine Adenovirus Type 3 Expressing Full-Length and Truncated Form of Bovine Herpesvirus Type 1 Glycoprotein gD

AbstractUsing the homologous recombination machinery ofE. coli,a 1.245-kb deletion was introduced in the E3 region of bovine adenovirus 3 (BAV3) genomic DNA cloned in a plasmid. Transfection of the restriction enzyme-excised, linear E3-deleted BAV3 genomic DNA into primary fetal bovine retina cells produced infectious virus (BAV3.E3d), suggesting that all the E3-specific open reading frames are nonessential for virus replicationin vitro. Using a similar approach, we constructed replication-competent (BAV3.E3gD and BAV3.E3gDt) BAV3 recombinant expressing full-length (gD) or truncated (gDt) glycoprotein of bovine herpes virus 1. Recombinant gD and gDt proteins expressed by BAV3.E3gD and BAV3.E3gDt, respectively, were recognized by gD-specific monoclonal antibodies directed against conformational epitopes, suggesting that antigenicity of recombinant gD and gDt was similar to that of the native gD expressed in bovine herpes virus 1-infected cells. Intranasal immunization of cotton rats induced strong gD- and BAV3-specific IgA and IgG immune responses. These results suggest that replication-competent bovine adenovirus 3-based vectors have potential for the delivery of vaccine antigens to the mucosal surfaces of animals

Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference

AbstractThe severe acute respiratory syndrome (SARS) has been one of the most epidemic diseases threatening human health all over the world. Based on clinical studies, SARS-CoV (the SARS-associated coronavirus), a novel coronavirus, is reported as the pathogen responsible for the disease. To date, no effective and specific therapeutic method can be used to treat patients suffering from SARS-CoV infection. RNA interference (RNAi) is a process by which the introduced small interfering RNA (siRNA) could cause the degradation of mRNA with identical sequence specificity. The RNAi methodology has been used as a tool to silence genes in cultured cells and in animals. Recently, this technique was employed in anti-virus infections in human immunodeficiency virus and hepatitis C/B virus. In this study, RNAi technology has been applied to explore the possibility for prevention of SARS-CoV infection. We constructed specific siRNAs targeting the S gene in SARS-CoV. We demonstrated that the siRNAs could effectively and specifically inhibit gene expression of Spike protein in SARS-CoV-infected cells. Our study provided evidence that RNAi could be a tool for inhibition of SARS-CoV

Peste des petits ruminants virus tissue tropism and pathogenesis in sheep and goats following experimental infection

Peste des petits ruminants (PPR) is a viral disease which primarily affects small ruminants, causing significant economic losses for the livestock industry in developing countries. It is endemic in Saharan and sub-Saharan Africa, the Middle East and the Indian sub-continent. The primary hosts for peste des petits ruminants virus (PPRV) are goats and sheep; however recent models studying the pathology, disease progression and viremia of PPRV have focused primarily on goat models. This study evaluates the tissue tropism and pathogenesis of PPR following experimental infection of sheep and goats using a quantitative time-course study. Upon infection with a virulent strain of PPRV, both sheep and goats developed clinical signs and lesions typical of PPR, although sheep displayed milder clinical disease compared to goats. Tissue tropism of PPRV was evaluated by real-time RT-PCR and immunohistochemistry. Lymph nodes, lymphoid tissue and digestive tract organs were the predominant sites of virus replication. The results presented in this study provide models for the comparative evaluation of PPRV pathogenesis and tissue tropism in both sheep and goats. These models are suitable for the establishment of experimental parameters necessary for the evaluation of vaccines, as well as further studies into PPRV-host interactions.A Canadian International Food Security Research Fund (CIFSRF) grant (no. 106930: Livestock vaccines against viral diseases for sub-Saharan Africa) by the Canadian International Development Research Centre (IDRC) and Canadian International Development Agency (CIDA).http://www.plosone.orgam201