Surveillance of Emerging Livestock Viruses

Accurate and rapid diagnostic tests are essential for the management and control of emerging infectious diseases. Therefore, the overall goals of studies presented in this dissertation were to provide improved diagnostic and surveillance capabilities for several emerging viral diseases impacting the livestock industry and to provide insight into appropriate control strategies. Recent, severe outbreaks of porcine epidemic diarrhea virus (PEDV) in Asia and North America highlighted the need for well-validated diagnostic tests for identification of PEDV infected animals and evaluation of their immune status. PEDV was first detected in the U.S. in May 2013 and another swine coronavirus, porcine deltacoronavirus (PDCoV) was identified in the U.S. in February 2014. Both pathogens spread rapidly across the U.S., severely impacting the swine industry. As part of this project, well-validated serological assays for the detection of antibodies against PEDV and PDCoV were developed. Monoclonal antibodies (mAbs) against selected PEDV and PDCoV structural proteins were also developed and applied to serological and antigen detection assays. Newly developed fluorescence-based virus neutralization assays also provide valuable tools for assessment of vaccine candidates or protective immunity. The PEDV spike (S) glycoprotein plays a key role in virus entry into target cells and mediates the functions of receptor binding and fusion during entry. Therefore, immunodominant neutralizing epitopes of PEDV were identified using a panel of S-specific mAbs. Epitope mapping by peptide ELISA revealed that seven mAbs recognized linear neutralizing epitopes located in the N-terminus of the S2 glycoprotein subunit. Two mAbs recognized a neutralizing epitope located in the C-terminus of S2, while only one neutralizing mAb reacted against a region of the S1 glycoprotein subunit. These results indicate that the S2 glycoprotein subunit contains major antigenic determinants and, perhaps, the immunodominant neutralizing epitopes of PEDV. Influenza D virus is a recently described virus that affects cattle and limited serological testing capability is currently available. Therefore, new ELISA-based serological assays based on expressed protein antigens representing conserved regions of the nucleoprotein and hemagglutinin-esterase proteins were developed. Results of these assays correlate well with hemagglutination inhibition (HI) and virus neutralization assays, providing new tools for ongoing sero-surveillance and control efforts

Development of an Indirect ELISA, Blocking ELISA, Fluorescent Microsphere Immunoassay and Fluorescent Focus Neutralization Assay for Serologic Evaluation of Exposure to North American Strains of Porcine Epidemic Diarrhea Virus

Recent, severe outbreaks of porcine epidemic diarrhea virus (PEDV) in Asia and North America highlight the need for well-validated diagnostic tests for the identification of PEDV infected animals and evaluation of their immune status to this virus. PEDV was first detected in the U.S. in May 2013 and spread rapidly across the country. Some serological assays for PEDV have been previously described, but few were readily available in the U.S. Several U.S. laboratories quickly developed indirect fluorescent antibody (IFA) assays for the detection of antibodies to PEDV in swine serum, indicating prior exposure. However, the IFA has several disadvantages, including low throughput and relatively subjective interpretation. Different serologic test formats have advantages and disadvantages, depending on the questions being asked, so a full repertoire of tests is useful. Therefore, the objective of this study was to develop and validate multiple improved serological assays for PEDV, including an indirect ELISA (iELISA); a highly specific monoclonal antibody-based blocking ELISA (bELISA); fluorescent microsphere immunoassays (FMIA) that can be multiplexed to monitor exposure to multiple antigens and pathogens simultaneously; and a fluorescent focus neutralization assay (FFN) to measure functional virus neutralizing antibodies

ANTIVIRAL AND IMMUNE STIMULANT ACTIVITIES OF GLYCYRRHIZIN AGAINST DUCK HEPATITIS VIRUS

This study was conducted to investigate the effect of glycyrrhizin as an immune stimulant against duck hepatitis virus (DHV). In vitro study was carried out to determine cytotoxic and antiviral effects of glycyrrhizin in VERO cells. In vivo study was performed on 40 one-day-old White Pekin ducklings. –and the birds weres divided into 4 groups: control, glycyrrhizin treated, vaccinated with live attenuated DHV vaccine and glycyrrhizin treated and vaccinated; to investigate the changes in immunity and challenge test. Blood samples were collected from each duckling for evaluation of cellular and humeral immunity. The in vitro results revealed that glycyrrhizin had antiviral and no toxic effects till 106 dilutions. Higher antibody titer was observed from the 5th week till the end of experiment in glycyrrhizin and vaccinated group. Treatment with glycyrrhizin alone or with DHV vaccine demonstrated a pronounced lymphocytic proliferation response after 4 days post-inoculation till the end of experiment, while vaccinated group revealed a pronounced proliferation response after 24 days post-inoculation. Treatment with glycyrrhizin alone or combination with DHV vaccine revealed good immune stimulant and antiviral effect against DHV

Potent Antiviral Activity of Vitamin B12 against Severe Acute Respiratory Syndrome Coronavirus 2, Middle East Respiratory Syndrome Coronavirus, and Human Coronavirus 229E

Repurposing vitamins as antiviral supporting agents is a rapid approach used to control emerging viral infections. Although there is considerable evidence supporting the use of vitamin supplementation in viral infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the specific role of each vitamin in defending against coronaviruses remains unclear. Antiviral activities of available vitamins on the infectivity and replication of human coronaviruses, namely, SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV), and human coronavirus 229E (HCoV-229E), were investigated using in silico and in vitro studies. We identified potential broad-spectrum inhibitor effects of Hydroxocobalamin and Methylcobalamin against the three tested CoVs. Cyanocobalamin could selectively affect SARS-CoV-2 but not MERS-CoV and HCoV-229E. Methylcobalamin showed significantly higher inhibition values on SARS-CoV-2 compared with Hydroxocobalamin and Cyanocobalamin, while Hydroxocobalamin showed the highest potent antiviral activity against MERS-CoV and Cyanocobalamin against HCoV-229E. Furthermore, in silico studies were performed for these promising vitamins to investigate their interaction with SARS-CoV-2, MERS-CoV, and HCoV-229E viral-specific cell receptors (ACE2, DPP4, and hAPN protein, respectively) and viral proteins (S-RBD, 3CL pro, RdRp), suggesting that Hydroxocobalamin, Methylcobalamin, and Cyanocobalamin may have significant binding affinity to these proteins. These results show that Methylcobalamin may have potential benefits for coronavirus-infected patients