We report a previously unrecognized complexity to the ecology of rabies in wildlife. Rabies-specific virus-neutralizing antibodies in spotted hyenas, the most numerous large carnivore in the Serengeti ecosystem (Tanzania, East Africa), revealed a high frequency of exposure of 37.0% to rabies virus, and reverse transcriptase (RT) PCR demonstrated rabies RNA in 13.0% of hyenas. Despite this high frequency, exposure neither caused symptomatic rabies nor decreased survival among members of hyena social groups monitored for 9 to13 years. Repeated, intermittent presence of virus in saliva of 45.5% of seropositive hyenas indicated a ''carrier'' state. Rabies isolates from Serengeti hyenas differed significantly (8.5% sequence divergence) from those isolated from other Serengeti carnivores, suggesting that at least two separate strains circulate within the Serengeti carnivore community. This finding is consistent with the fact that exposure in hyenas increased with age and social status, following a pattern predicted by intraspecific age and social-status-dependent oral and bite contact rates. High seroprevalence of rabies, low basic reproductive rate of the virus (R0) of 1.9, a carrier state, and the absence of symptomatic rabies in a carnivore in an ecosystem with multihost and multistrain maintenance has not been previously demonstrated for rabies. Because of the substantial differences between the hyena viral isolates and those from canids and viverrids in the Serengeti, it is unlikely that spotted hyenas were the source of rabies virus that killed several African wild dog packs in the Serengeti ecosystem in the 1990s.

The present study describes the generation of a new Orf virus (ORFV) recombinant, D1701-V-RabG, expressing the rabies virus (RABV) glycoprotein that is correctly presented on the surface of infected cells without the need of replication or production of infectious recombinant virus. One single immunization with recombinant ORFV can stimulate high RABV-specific virus-neutralizing antibody (VNA) titers in mice, cats, and dogs, representing all nonpermissive hosts for the ORFV vector. The protective immune response against severe lethal challenge infection was analyzed in detail in mice using different dosages, numbers, and routes for immunization with the ORFV recombinant. Long-term levels of VNA could be elicited that remained greater than 0.5 IU per ml serum, indicative for the protective status. Single applications of higher doses (10 7 PFU) can be sufficient to confer complete protection against intracranial (i.c.) challenge, whereas booster immunization was needed for protection by the application of lower dosages. Anamnestic immune responses were achieved by each of the seven tested routes of inoculation, including oral application. Finally, in vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T cell subpopulations during immunization and/or challenge infection attested the importance of CD4 T cells for the induction of protective immunity by D1701-V-RabG. This report demonstrates another example of the potential of the ORFV vector and also indicates the capability of the new recombinant for vaccination of animals.

Influenza, a respiratory disease caused by influenza viruses, still represents a major threat to humans and several animal species. Besides vaccination, only two classes of drugs are available for antiviral treatment against this pathogen. Thus, there is a strong need for new effective antivirals against influenza viruses. Here, we tested Ladania067, an extract from the leaves of the wild black currant (Ribes nigrum folium) for potential antiviral activity against influenza A virus in vitro and in vivo. In the range of 0–1 mg/ml the extract showed no cytotoxic effect on three cell lines and a CC50 of 0.5 ± 0.3 mg/ml, on peripheral blood mononuclear cells. Furthermore, the extract did not influence the proliferative status of human lymphocytes. In contrast, Ladania067 was highly effective (EC50 value: 49.3 ± 1.1 ng/ml) against the human pandemic influenza virus strain A/Regensburg/D6/09 (H1N1). The extract exhibited an antiviral effect when the virus was pre-incubated prior to infection or when added directly after infection. No antiviral effect was found when infected cells were treated 2, 4, or 8 h after infection, indicating that Ladania067 blocks a very early step in the virus infection cycle. In the mouse infection model we were able to demonstrate that an intranasal application of 500 μg Ladania067 inhibits progeny virus titers in the lung up to 85% after 24 h. We conclude that the extract from the leaves of the wild black currant may be a promising source for the identification of new molecules with antiviral functions against influenza virus.