The molecular epidemiology of bluetongue virus in northern Colorado

Abstract

Economic losses due to trade restrictions based on BTV have been estimated to exceed $125 million U.S. per year in the U.S., and have been estimated to exceed $3 billion per year worldwide. Despite many years of study, the overwintering mechanism of bluetongue virus (BTV) has never been elucidated to a scientifically satisfactory degree. This is an important area of research because current trade agreements between the United States and its partners allow for the regionalization of disease entities (and their import/export restrictions) based on adequate scientific knowledge of the epidemiology of the disease. Studies in this dissertation were guided by the hypothesis that BTV overwinters in its arthropod vector, Culicoides sonorensis. The overwintering mechanism of BTV was investigated using a variety of traditional virologic methods as well as current molecular methods. In toto, the studies were designed to provide a clear picture of the epidemiology of BTV on a microgeographic scale, thereby complementing other studies on a macrogeographic scale. Virus isolates were obtained from the USD A-Arthropod Bome Animal Diseases Research Laboratory (ABADRL) and from collections at the Arthropod-borne and Infectious Diseases Laboratory at Colorado State University (AIDL). Field isolates were characterized by phylogenetic analysis of sequence data from three genome segments (L2, S7, and S10). The viral isolates showed a degree of genetic identity inconsistent with other hypotheses concerning the overwintering mechanism of BTV. In addition, RT-PCR and Northern blot analysis of total RNA from cell-culture models of natural infection, using both mammalian cell lines and insect cells obtained from ABADRL, were performed to characterize a possible mechanism of persistence of the virus in insect cells. Viral nucleic acid was detected in presumably uninfected cell cultures, as well as in field-collected culicoid larvae by RT-PCR, but not by Northern blot analysis. The previous target of field diagnostics (L2, which encodes the major antigenic determinant VP2) was not detected in insect cell cultures or field-collected larvae; this has obvious implications to the "diagnosis" of endemic and BTV-free regions of the country, as well as surveillance programs based on invertebrate collections.