Dissertation - Open Access
Doctor of Philosophy (PhD)
Department / School
Biology and Microbiology
Radhey S Kaushik
guinea pigs, Hemagglutinin esterase fusion, influenza C, influenza D virus, pathogenesis, primary respiratory epithelial cells
Influenza D virus (IDV) is a novel influenza virus that infects cattle and swine, with cattle as its primary host species. The goal of our first study was to investigate the replication and transmission of bovine IDV in guinea pigs. Following direct intranasal inoculation of animals, the virus was detected in nasal washes of infected animals during the first 7 days post-infection. High viral titers were obtained from nasal turbinates and lung tissues of directly inoculated animals. Further, bovine IDV was able to transmit from the infected guinea pigs to sentinel animals by means of contact and not by aerosol dissemination under the experimental conditions tested in this study. Despite exhibiting no clinical signs, infected guinea pigs developed seroconversion and the viral antigen was detected in lungs of animals by immunohistochemistry. The observation that bovine IDV replicated in the respiratory tract of guinea pigs was similar to observations described previously in studies of gnotobiotic calves and pigs experimentally infected with bovine IDV but different from those described previously in experimental infections in ferrets and swine with a swine IDV, which supported virus replication only in the upper respiratory tract and not in the lower respiratory tract, including lung. Our study established that guinea pigs could be used as an animal model for studying this newly emerging influenza virus. Influenza D virus isolated from the cattle and swine populations from North America and Eurasia shares 50% homology to the human influenza C virus. The goal of our second study was to investigate the replication kinetics and virulence of bovine and swine influenza D isolates (96-98% homology), in comparison to human influenza C in guinea pigs. Despite the similarity, both bovine and swine IDVs differ antigenically and genetically and belong to two different lineages. Guinea pigs upon intranasal inoculation of D/bovine/660/Oklahoma/2013 (bovine IDV), D/swine/1334/Oklahoma/2011 (swine IDV) and C/ Victoria/2012 (human ICV) did not exhibit any clinical signs. However, all the infected animals seroconverted at 7 days post-infection (dpi). Guinea pigs infected with ICV did not shed the virus in nasal washes at 1 dpi and only 2/8 shed virus at 3 dpi. In contrast, in bovine IDV infected group, 9/10 animals shed the virus in nasal washes at 1 dpi, while the swine IDV group (8/8) began to shed the virus only at 3 dpi. Hence, the disparity in the virus-shedding pattern of swine IDV could be an adaptation lag due to the subtle difference in receptor binding specificity and virus tropism. Deep RNA sequencing of viral genomes in the nasal washes, receptor binding preference, and structural modeling of receptor binding domain of hemagglutinin-esterase fusion protein are currently underway to identify the key factors and mechanisms involved in the differential replication kinetics, viral tropism, pathogenesis of the bovine and swine influenza D viruses. Further, our third project was aimed at developing a good primary culture system from swine for studying the virulence and pathogenesis. Influenza viruses are a group of respiratory pathogens that have evolved into four different types: A, B, C, and D. One common feature is that all four types are capable of replication and transmission among pigs. Human respiratory primary epithelial cell culture has been recently utilized to examine the replication and pathogenesis of influenza A viruses. However, little has been made in the development of the autologous cell culture system from swine to study influenza viruses. Here we describe the development of primary epithelial cells from swine nasal turbinates, trachea and lungs and determine their utility in the replication of four types of influenza viruses. Phenotypic characterization using immunocytochemistry coupled with flow cytometry analysis showed that cytokeratin was expressed at high levels in swine nasal turbinates, trachea, and lung cells, while the relatively low abundance of other epithelial cell markers (desmin, α-SMA, and vimentin) was detected. In addition, all three swine cells were found able to undergo the polarization as measured by trans-epithelial electrical resistance (TEER) and expression of tight junction proteins including claudin-1, -3, Zona occludens protein -1 (ZO-1) and occludin-1. These results strongly suggest that the developed swine primary cells possess common characteristics of epithelial cells. Furthermore, sialic acid receptor profile analysis through lectin binding assay with Sambucus Nigra Lectin (SNA) and Maackia Amurensis Lectin II (MAL-II) demonstrated that three swine primary epithelial cells expressed higher levels of alpha 2,6 linkage sialic acid (SNA) than alpha 2,3 linkage sialic acid receptors (MAL-II). Finally, all three primary cells supported the replication of Influenza A, B, C and D viruses to an appreciable level, but virus type-dependent replication kinetics were observed. Overall, these swine respiratory primary cells showed epithelial phenotype and are suitable for studying the comparative biology and pathobiology of four types of influenza viruses.
Library of Congress Subject Headings
Influenza viruses -- Reproduction.
Guinea pigs as laboratory animals.
Includes bibliographical references
Number of Pages
South Dakota State University
Sreenivasan, Chithra Chembil, "Replication, Virulence, and Pathogenesis of Influenza Viruses" (2018). Electronic Theses and Dissertations. 2654.