Experimental infection of small ruminants with bluetongue virus expressing Toggenburg Orbivirus proteins.

Bluetongue virus (BTV) is the prototype orbivirus (Reoviridae family, genus Orbivirus) consisting of more than 24 recognized serotypes or neutralization groups. Recently, new BTV serotypes in goats have been found; serotype 25 (Toggenburg Orbivirusor TOV), serotype 26 (KUW2010/02), and serotype 27 from Corsica, France. KUW2010/02 has been isolated in mammalian cells but is not replicating in Culicoides cells. TOVhas been detected in goats but could not been cultured, although TOV has been successfully passed to naïve animals by experimental infection using viremic blood. Genome segments Seg-2[VP2], Seg-6[VP5], Seg-7[VP7], and Seg-10[NS3/NS3a] expressing the respective TOV proteins were incorporated in BTV using reverse genetics, demonstrating that these TOV proteins are functional in BTV replication. Depending on the incorporated TOV proteins, in vitro replication is, however, decreased compared to the ancestor BTV, in particular by TOV-VP5. Sheep and goats were experimentally infected with BTV expressing both outer capsid proteins VP2 and VP5 of TOV, so-named 'TOV-serotyped BTV'. Viremia was not detected in sheep, and hardly detected in goats after infection with TOV-serotyped BTV. Seroconversion by cELISA, however, was detected, suggesting that TOV-serotyped BTV replicates in small ruminants. One goat was coincidentally pregnant, and the fetus was strong PCR-positive in blood samples and several organs, which conclusively demonstrates that TOV-serotyped BTV replicates in vivo.

Long-term infection of goats with bluetongue virus serotype 25.

Toggenburg Orbivirus (TOV) is the prototype of bluetongue virus serotype 25 (BTV-25). It was first detected in goats in Switzerland in 2008. The virus does not induce clinical signs in infected goats. In field samples viral RNA could be detected only in goats and never in other ruminants. BTV-25 RNA was repeatedly detected for more than one year in the blood of goats from a single flock in Principality of Liechtenstein. Since viral persistence over such a long period has never been reported for bluetongue, blood samples from 110 goats and 2 sheep of that flock were collected during a period of up to two years and analyzed for the presence of BTV-25 RNA and antibodies. Most of the animals which tested positive for BTV-25 RNA, remained positive during the whole investigation period. Moreover, five of these goats were BTV-25 RNA positive over a period of 19-25 months. A weak antibody response against BTV VP7 was commonly observed. As BTV-25 cannot be propagated in any culture system, the presence of virus could only be demonstrated in samples by viral RNA detection using RT-qPCR. To address the question of infectivity of the virus in blood from long-term positive animals, goats were experimentally infected with this blood. Viral replication was demonstrated by increasing RNA amounts. Thus, our findings provide evidence that BTV-25 can persist much longer in an infected host than known so far for other BTV serotypes. Hence, persistence of infectious BTV represents an additional important factor in BTV epidemiology.