Mechanisms controlling the infection of Culicoides biting midges with bluetongue virus
The mechanisms controlling the transmission of bluetongue virus (DTV) by vector Culicoides species were studied using immunohistochemistry, virus titration assays, in vitro transmission tests, viral binding protein analyses and transmission electron microscopy. After infection with BTV by intrathoracic (IT) inoculation, 100% of C. variipennis individuals from a susceptible colony developed a fully disseminated infection and transmitted the virus through their saliva. However only 35.4% of midges were . persistently infected after ingestion of an infectious blood meal, while only 12.1 % of persistently infected midges transmitted the virus through their saliva. The titres of BTV were about 10,·oTCIDsJmidge [Standard error of means (SEM) of log-transformed data=0.15, n=1400] in IT inoculated midges and varied from 0.32 to lQs.oTCIDsJmidge in orally infected individuals. Only those midges containing ~1 03.oTCIDso of BTV could transmit the virus through their saliva. The following patterns were observed in orally (persistently) infected individuals: 1) virus was restricted to the anterior and posterior midgut, and the foregut-midgut junction; 2) virus replicated in the gut cells, disseminated into the haemocoel but could only be detected in a few sporadic fat body cells beyond the gut; 3) virus escaped from the gut cells into the haemocoel and replicated in some secondary organs/tissues but at low levels; 4) a fully disseminated infection was observed and virus replicated in the haemocoel and secondary organs/tissues, including the salivary glands, at high levels. The infection of the gut can be divided into two main types: 1) virus replication in gut cells ranging from very low to higher levels but with virus spread throughout the cytoplasm of the infected cells; 2) virus positive reaction restricted to endosome-like structures in the cytoplasm of some gut cells. BTV was detected in the anterior and posterior midgut, foregut-midgut junction, fat body, ganglia, salivary glands and ommatidia of the compound eyes of some infected midges. No virus was ever found in the hindgut cells, muscles, Malpighian tubes and oocytes/nurse cells of the ovaries. BTV infection of the salivary glands of C. l'ariipcnnis was shown to follow a typical pattern. Virus entered the acinar cells from the haemococl passing through the basement membrane, then localised and replicated in virus inclusion bodies (VIBs) in the cytoplasm of acinar cells. Mature progeny virus particles were released into acini, then transported through intermediate ducts and accumulated in crystalline arrays in the lumen of the major secretory ducts. No virus was released back into the haemocoel through the basement membrane; nor was virus released back into acinar cells from the acini. Nervous tissue of C. l'ariipennis is one of the most susceptible tissues to BTV. Ultrastructural observation showed characteristics ofBTV replication, including formation of VIBs, large amounts of progeny virus particles and tubules, in infected thoracic ganglia. A 60-kD viral protein adhered to both BHK-21 (mammalian) cells and a Culicoides cell line, KC cells. A 44-kD BTV viral protein, co-migrating with non structural protein NS2, adsorbed to BHK-21 cells but not to KC cells, while a 39.6 kD viral protein, co-migrating with major inner capsid protein VP7, adhered only to KC cells but not to BHK-21 cells.