Browsing by Subject "Virus"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Concentration, size distribution, and control of swine viruses associated with airborne particles
    (2016-03) Alonso Garcia-Mochales, Carmen
    Infectious diseases that affect swine represent a threat to global food security. Some of these diseases are zoonotic and also represent a risk to public health. Among all infectious agents affecting swine, airborne pathogens are especially challenging since they are difficult to contain and control both within farms and regionally. Infectious agents travel through the air associated with particles of variable composition and size. Particle size influences the likelihood that infectious agents will be transmitted to susceptible hosts. This research focuses on the quantification and size distribution of swine viruses associated with airborne particles, and on the efficacy of a novel technology for reducing airborne particles.
  • Thumbnail Image
    Item
    Detection, diagnostics, and characterization of virus-like organisms and conformational disease-like proteins in plants
    (2016-10) Bratsch, Sara Ann
    The projects in this thesis all investigated virus-like organisms in agronomically important plants. Chapter one consists of reports describing viruses identified in new hosts or locations. Tobacco rattle virus (TRV) was identified in symptomatic Phryma leptostachya L, a native perennial, from plants in an uncultivated habitat and suggests that TRV may be endemic to North America. Since TRV is the causal organism of corky ringspot of potato this study raises the possibility that native perennial plants could serve as a potential reservoir to cause disease in potato. Canna yellow mottle virus was isolated for the first time from symptomatic Canna indica in Kenya. Cut flowers are a major agronomic crop of Kenya and growers should plant only virus indexed plants to limit losses from virus infection. Orchid fleck virus was confirmed by microscopy and sequence analysis for the first time in the United States in Phalaenopsis hybrida. Asymptomatic P. hybrida tested by one step reverse transcription polymerase chain reaction (RT-PCR) did not yield the expected product while a two step RT-PCR, creating cDNA first, yielded the expected product. This indicates the one step RT-PCR diagnostic test can yield false negatives for asymptomatic plants. Chapter two describes the production of polyclonal antibodies for the detection of Orchid fleck virus (OFV). OFV is a mite transmitted virus and has been reported word wide. The previous project identified a need for a reliable, inexpensive method to detect OFV in plants use for propagation, breeding, conservatories, or virus indexing projects. Polyclonal antibodies were produced in rabbits against Escherichia coli expressed OFV phosphoprotein and matrix protein. The resulting antiserums were assayed in PTA-ELISA and DAS-ELISA. OFV phosphoprotein antisera in PTA-ELISA readily differentiated between healthy and OFV infected orchid (Phalaenopsis hybrida) tissue. OFV matrix antisera in PTA-ELISA detected bacterially-expressed protein but did not differentiate between healthy or OFV infected tissue. The OFV phosphoprotein antiserum can be used by in PTA-ELISA to reliably detect OFV. Chapter three describes the molecular and biological characterization of a new Nepovirus causing a leaf mottling disease in Petunia hybrida. The sequence of the majority of the genome was determined by next generation sequencing and the sequence of remainder of the genome was obtained using a 5’ RACE amplification and RT-PCR using poly-A tail and virus specific primers. Due to phylogenetic relationship and sufficient genome dissimilarity to characterized viruses I propose the name of Petunia Chlorotic Mottle Virus for a new Nepovirus. The fourth chapter describes characterization of filamentous virus-like particles in members of the Asteraceae plant family including sunflower, chrysanthemum, coneflower, gerbera daisy, and zinnia. The filaments were 7-10 nanometers in diameter and could exceed 3,000 nm in length. The N-terminal sequences of the major proteins associated with purified filaments from several species were nearly identical and shared homology with the kunitz soybean trypsin inhibitor (KTI) family of proteins. CID MS/MS sequencing of the major proteins of purified sunflower filaments also shared homology with a KTI sequence. A Western blot using antiserum prepared against recombinant sunflower KTI protein labeled the observable protein bands from sunflower filaments. Filaments composed of a major protein of KTI have been found across the Asteraceae family but have not been observed in dandelion, thistle, or lettuce.
  • Thumbnail Image
    Item
    Improved diagnosis and management of viral hemorrhagic septicemia virus in fish
    (2013-02) Phelps, Nicholas
    Viral hemorrhagic septicemia virus (VHSV) is a highly contagious and pathogenic virus, affecting more than 70 farm raised and wild fish species worldwide. A new viral strain (VHSV-IVb) has proven both virulent and persistent, spreading throughout the Great Lakes of North America and to inland water bodies in the region. As this new biological hazard continues to grow, so too must our understanding of the disease. The focus of this dissertation is to improve diagnostic capacity and management of VHSV-IVb to rapidly respond to outbreaks, prevent further dissemination, and scientifically justify current prevention strategies. To better understand the geographic distribution of the virus, we used a modified real time reverse transcription-polymerase chain reaction (rRT-PCR) assay for high-throughput testing of fish for VHSV. The assay was shown to be twice as sensitive as the gold standard, virus isolation, and did not cross react with other viruses found in fish. In addition, the diagnostic turnaround time was reduced from 28-30 days for virus isolation to 2-4 days for rRT-PCR. To demonstrate the usefulness of the rRT-PCR assay, 115 high priority water bodies in Minnesota were tested by both methods from April 2010 - June 2011. All survey sites tested negative for VHSV by both methods. The survey results have informed fisheries managers on the absence of VHSV in Minnesota and have better prepared them for the eventual arrival of the disease. In addition, the results demonstrate the value of this rRT-PCR as a surveillance tool to rapidly identify an outbreak so that it can be controlled in a timely manner. The aforementioned rRT-PCR assay (Phelps et al. 2012) along with another (Jonstrup et al. 2012), were evaluated for the potential for sample-induced inhibition from common diagnostic samples, including kidney/spleen, entire viscera, and ovarian fluid. The detection of high, medium, and low VHSV-IVb dilutions in each tissue type was not affected using the assay by Jonstrup et al (2012). However, using the assay by Phelps et al. (2012), the detection of VHSV-IVb was decreased for the kidney/spleen samples spiked with low virus levels and increased for the ovarian fluid spiked with medium virus levels. Entire viscera, the tissue type most likely to inhibit the rRT-PCR reaction, did not affect the sensitivity of virus detection for either assay. The emergence of VHSV-IVb in the Great Lakes region has resulted in unprecedented regulatory response to better manage the disease. In Minnesota, all VHSV-susceptible fish must be inspected annually prior to intra or interstate movement - a significant bottleneck for the aquaculture industry. In 2009, Minnesota enacted legislation requiring fish for regulatory health inspections to be collected by a Minnesota Department of Natural Resources (MNDNR) approved individual. The so-called Fish Health Collector could be 1) an American Fisheries Society - Fish Health Section Fish Health Inspector or Pathologist, 2) an accredited veterinarian with approved training, or 3) an individual (i.e. MNDNR field biologist) with approved training. In response, a fish health collector training workshop was developed for veterinarians and field biologists to fulfill the MNDNR requirements. A manual was developed to supplement a full-day workshop and provide the basic information and references to perform a fish health collection. This training resulted in a sufficient number of fish health collectors, well distributed across Minnesota, now available to rapidly respond to a disease outbreak and better serve the regulatory needs of the aquaculture industry. In the USA, current state and federal fish health regulations target the spread of VHSV-IVb through movement restrictions of live fish but largely ignore the potential for the virus to be spread through the commercial distribution and use of frozen baitfish from VHSV-IVb positive regions. Some state laws do require treatment of frozen baitfish to inactivate VHSV and additional methods have been proposed, but there have been few scientific studies examining the efficacy of these treatments. In an effort to evaluate these treatments, bluegills were challenged with VHSV-IVb, frozen to represent standard industry methods, disinfected by various treatments, and tested for infectious VHSV-IVb using virus isolation. The virus was isolated from 70% of fish subjected to three freeze thaw cycles. All other treatment methods were effective in inactivating the virus, including treatment with isopropyl alcohol, mineral oil, salt with borax, and dehydration. Dehydration followed by rehydration is rapid and effective, and therefore, seems to be the best option for inactivating VHSV-IVb present in frozen baitfish while maintaining their usefulness as bait. Monitoring or regulating all risk factors for the transmission of VHSV is an infeasible task. A semi-quantitative risk assessment model was utilized to focus VHSV management efforts in Minnesota. The risk of VHSV introduction to major watersheds in Minnesota was directly correlated with proximity to Lake Superior, the only VHSV-positive waterbody in the state. Although the current regulations are uniform across Minnesota, the risk varied for specific locations within the watersheds. For example, the introduction of game fish for stock enhancement (a common fisheries management practice) was found to be a significant risk factor for VHSV introduction into public waterbodies and waterbodies frequently used for wild baitfish harvest. Aquaculture facilities with strict biosecurity programs and frequent health inspections received the lowest risk scores and were largely considered protected and of low risk for VHSV introduction. These results suggest the current management strategy, based on political boundaries, should be reevaluated. A risk-based management strategy would better allocate efforts to watersheds or specific waterbodies at higher risk and relax efforts in areas of lower risk of VHSV introduction in Minnesota.