In Vitro Expression of Full-Length and Truncated Bovine Respiratory Syncytial Virus G Proteins and Their Antibody Responses in BALB/c Mice

Bovine respiratory syncytial virus (BRSV) is a primary cause of lower respiratory tract disease in calves. Protection is incomplete following vaccination or natural infection, as re-infections are common. The objectives of this study were to create plasmid DNA constructs encoding the full-length, secreted, or conserved region of the BRSV G glycoprotein, and to compare and evaluate their expression in cell culture and potential to induce antibody responses in BALB/c mice. Transfection of COS-7 cells with plasmid DNA resulted in expression of the BRSV G region from each of the plasmid DNA constructs. Following inoculation of BALB/c mice with plasmid DNA, a significant and equivalent anti-BRSV G IgG response was elicited to the full-length and truncated BRSV G proteins. These constructs may be used to study host pathological and immunological responses

Experimental infection of conventional nursing pigs and their dams with \u3ci\u3ePorcine deltacoronavirus\u3c/i\u3e

Porcine deltacoronavirus (PDCoV) is a newly identified virus that has been detected in swine herds of North America associated with enteric disease. The aim of this study was to demonstrate the pathogenicity, course of infection, virus kinetics, and aerosol transmission of PDCoV using 87 conventional piglets and their 9 dams, including aerosol and contact controls to emulate field conditions. Piglets 2–4 days of age and their dams were administered an oronasal PDCoV inoculum with a quantitative real-time reverse transcription (qRT)-PCR quantification cycle (Cq) value of 22 that was generated from a field sample having 100% nucleotide identity to USA/Illinois121/2014 determined by metagenomic sequencing and testing negative for other enteric disease agents using standard assays. Serial samples of blood, serum, oral fluids, nasal and fecal swabs, and tissues from sequential autopsy, conducted daily on days 1–8 and regular intervals thereafter, were collected throughout the 42-day study for qRT-PCR, histopathology, and immunohistochemistry. Diarrhea developed in all inoculated and contact control pigs, including dams, by 2 days post-inoculation (dpi) and in aerosol control pigs and dams by 3–4 dpi, with resolution occurring by 12 dpi. Mild to severe atrophic enteritis with PDCoV antigen staining was observed in the small intestine of affected piglets from 2 to 8 dpi. Mesenteric lymph node and small intestine were the primary sites of antigen detection by immunohistochemistry, and virus RNA was detected in these tissues to the end of the study. Virus RNA was detectable in piglet fecal swabs to 21 dpi, and dams to 14–35 dpi

VBMS 403: Integrated Principles and Prevention of Livestock Diseases

This portfolio documents the teaching objectives, strategies, assessments, and changes implemented for VBMS 403: Integrated Principles and Prevention of Livestock Diseases, an Achievement-Centered Education (ACE) 10 Capstone course taught during the Spring Semester through the School of Veterinary Medicine and Biomedical Sciences at the University of Nebraska-Lincoln. Teaching methods and course activities included traditional lecturing with quizzes and examinations, in-class discussions, short reading assignments, and the generation of a scholarly term paper demonstrating broad knowledge, technical proficiency, information collection, synthesis, interpretation, and presentation. This portfolio documents the qualitative and quantitative methods used to assess the course learning objectives (goals). My participation in the Peer Review of Teaching Project was aimed at improving as an instructor in the classroom, demonstrating my commitment to my position and gaining a better understanding of pedagogy. The information and instruction gained by the preparation of this benchmark portfolio was valuable and will be used in my other courses

Detection and Quantitation of Bovine Respiratory Syncytial Virus Using Real-Time Quantitative RT-PCR and Quantitative Competitive RT-PCR Assays

A single tube, fluorogenic probe-based, real-time quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assay was developed for detection and quantitation of bovine respiratory syncytial virus (BRSV) using BioRad’s iCycler iQ™. Real-time Q-RT-PCR was compared with quantitative competitive RT-PCR (QC-RT-PCR) and viral titers. Viral mRNA levels were measured in BRSV-infected bovine turbinate cell lysate harvested at eight time points (1.5, 6, 12, 24, 36, 48, 60, 72 h) post-infection. A homologous BRSV cRNA standard was used for quantitation of the mRNA by plotting a standard curve of cycle threshold (Ct) values versus standard 10-fold dilutions of cRNA of known concentrations. Detection as low as 171 copies/μl of standard BRSV cRNA was possible. For QC-RT-PCR, a competitor RNA molecule having a deletion was designed and used for quantitation of the BRSV viral mRNA. The results of real-time Q-RT-PCR and QC-RT-PCR assays showed a positive correlation. Real-time Q-RT-PCR was a sensitive, specific, rapid, and efficient method that eliminates the post-PCR processing steps when compared to QC-RT-PCR. Quantitation of BRSV using real-time Q-RT-PCR will have application in studies aimed at understanding the pathogenesis of BRSV

Long-Term Clinicopathological Characteristics of Alpacas Naturally Infected with Bovine Viral Diarrhea Virus Type Ib

Background: Substantial bovine viral diarrhea virus (BVDV)-related production losses in North American alpaca herds have been associated with BVDV type Ib infection. Objectives: To classify and differentiate the long-term clinicopathological characteristics of BVDV type Ib infection of al- paca crias, after natural virus exposure. We hypothesized that persistently infected (PI) alpacas specifically demonstrate growth retardation, clinicopathological evidence of opportunistic infections, and early mortality. Animals: Thirty-five crias naturally exposed to BVDV (18 acute, 3 chronic, 14 PIs), and 19 healthy cohort controls of 5 northeastern alpaca farms were prospectively evaluated over 2 years (September 2005–September 2008). Methods: Observational cohort-control study. Results: Chronically (viremia 43 weeks) and PI crias demonstrated significantly lower birth weights, decreased growth rates, anemia, and monocytosis compared with control animals. Common clinical problems of PI alpacas included chronic wasting, diarrhea, and respiratory disease. Median survival of PI alpacas that died was 177 days (interquartile range, 555) with a case fatality rate of 50% within 6 months of life. Transplacental infection was confirmed in 82% (9/11) of pregnant females on 1 farm, resulting in the birth of 7 PI crias (7/10 deliveries; 1 animal was aborted). Mean gestation at the beginning and end of BVDV exposure was 64 and 114 days, respectively. Conclusions and Clinical Importance: Natural BVDV type 1b infection during early pregnancy resulted in a high incidence of PI offspring. Although PI alpacas may have distinct clinical characteristics, verification of persistent viremia in the absence of endogenous, neutralizing antibodies is essential to differentiate persistent from chronic infection

Influence of bovine respiratory syncytial virus F glycoprotein \u3ci\u3eN\u3c/i\u3e-linked glycans on in vitro expression and on antibody responses in BALB/c mice

Bovine respiratory syncytial virus (BRSV) is an etiological component of the bovine respiratory tract disease complex. Infection with BRSV following vaccination, or re-infection following natural infection is common since protection is incomplete. The objectives of this study were to create plasmid DNA constructs encoding single or multiple N-glycosylation-site deletion BRSV fusion (F) proteins, and evaluate their expression in cell culture, and potential to induce anti-BRSV F antibody responses in BALB/ c mice. Four plasmid DNAs were constructed, each encoding 1-4 N-glycosylation-site deletions: Gly4, Gly2/4, Gly1/2/4 and Gly1/2/3/4. Each of the N-glycosylation-site deletion BRSV F proteins were expressed in COS-7 cells following transfection with plasmid DNA. Inoculation of BALB/c mice with plasmid DNA, resulted in a significant anti-BRSV F IgG response to the wildtype (WT) F and glycosylation-site deletion protein Gly2/4. Gly2/4 elicited a higher antibody titer than the fully glycosylated WT F protein. Significant neutralizing antibody titers were detected following immunization with the Gly2/4 plasmid DNA. These glycosylation-site deletion BRSV F proteins will be useful to characterize the effects of glycosylation on immunogenicity in the natural host, and may lead to a new approach for the generation of BRSV vaccines

A Replicon \u3ci\u3eTrans\u3c/i\u3e-Packaging System Reveals the Requirement of Nonstructural Proteins for the Assembly of Bovine Viral Diarrhea Virus (BVDV) Virion

A selective trans-packaging system was developed to produce and isolate bovine viral diarrhea virus (BVDV) pseudo-particles with complementing reporter replicons and their packaging proteins expressed in trans with recombinant vaccinia virus. The encapsidation of replicon rNS3-5B was dependent not only on the in trans expression of structural proteins C, Erns, E1 and E2, but also the nonstructural proteins, p7 and contiguous precursor NS2-3-4A. Nonstructural p7, NS4B, NS5A or NS5B could be expressed in cis and in trans with precursor NS2-3-4A without significantly affecting virion assembly efficiency. NS2-3-4A was identified as an in trans functional precursor in virion assembly. BVDV genomes with mutant NS5B, which did not undergo active replication, were packaged 5-fold less efficiently than the intact genomes demonstrating the importance of replication in virion packaging. These results suggest that genome replication and assembly are closely associated, consistent with a model in which these two steps are coupled for maximum efficiency

Experimental infection of conventional nursing pigs and their dams with \u3ci\u3ePorcine deltacoronavirus\u3c/i\u3e

Porcine deltacoronavirus (PDCoV) is a newly identified virus that has been detected in swine herds of North America associated with enteric disease. The aim of this study was to demonstrate the pathogenicity, course of infection, virus kinetics, and aerosol transmission of PDCoV using 87 conventional piglets and their 9 dams, including aerosol and contact controls to emulate field conditions. Piglets 2–4 days of age and their dams were administered an oronasal PDCoV inoculum with a quantitative real-time reverse transcription (qRT)-PCR quantification cycle (Cq) value of 22 that was generated from a field sample having 100% nucleotide identity to USA/Illinois121/2014 determined by metagenomic sequencing and testing negative for other enteric disease agents using standard assays. Serial samples of blood, serum, oral fluids, nasal and fecal swabs, and tissues from sequential autopsy, conducted daily on days 1–8 and regular intervals thereafter, were collected throughout the 42-day study for qRT-PCR, histopathology, and immunohistochemistry. Diarrhea developed in all inoculated and contact control pigs, including dams, by 2 days post-inoculation (dpi) and in aerosol control pigs and dams by 3–4 dpi, with resolution occurring by 12 dpi. Mild to severe atrophic enteritis with PDCoV antigen staining was observed in the small intestine of affected piglets from 2 to 8 dpi. Mesenteric lymph node and small intestine were the primary sites of antigen detection by immunohistochemistry, and virus RNA was detected in these tissues to the end of the study. Virus RNA was detectable in piglet fecal swabs to 21 dpi, and dams to 14–35 dpi