The straw-coloured fruit bat (Eidolon helvum) is widespread in sub-Saharan Africa and is widely hunted for bushmeat. It is known to harbour a range of paramyxoviruses, including rubuloviruses and henipaviruses, but the zoonotic potential of these is unknown. We previously found a diversity of paramyxoviruses within a small, captive colony of E. helvum after it had been closed to contact with other bats for five years. In this study, we used under-roost urine collection to further investigate the paramyxovirus diversity and ecology in this colony, which had been closed to the outside for ten years at the time of sampling. By sampling urine weekly throughout an entire year, we investigated possible seasonal patterns of shedding of virus or viral RNA. Using a generic paramyxovirus L-gene PCR, we detected eight distinct paramyxovirus RNA sequences. Six distinct sequences were detected using a Henipavirus-specific PCR which targeted a different region of the L-gene. Sequence detection had a bi-annual pattern, with the greatest peak in July, although different RNA sequences appeared to have different shedding patterns. No significant associations were detected between sequence detection and birthing season, environmental temperature or humidity, and no signs of illness were detected in any of the bats in the colony during the period of sample collection.

The protein C is a small viral protein encoded in an overlapping frame of the P gene in the sub-family Orthoparamyxovirinae. This protein, expressed by alternative translation initiation, is a virulence factor that regulates viral transcription, replication and production of defective interfering RNA, interferes with the host-cell innate immunity systems and supports assembly of viral particles and budding. We expressed and purified full-length and an N-terminally truncated C protein from Tupaia paramyxovirus (TupV) C protein (genus Narmovirus). We solved the crystal structure of the C-terminal part of TupV C protein at a resolution of 2.4 Å and found that it is structurally similar to Sendai virus C protein, suggesting that despite undetectable sequence conservation, these proteins are homologous. We characterized both truncated and full-length proteins by SEC-MALLS and SEC-SAXS and described their solution structures by ensemble models. We established a minireplicon assay for the related Nipah virus (NiV) and showed that TupV C inhibited the expression of NiV minigenome in a concentration-dependent manner as efficiently as NiV C protein. A previous study found that the Orthoparamyxovirinae C proteins former two clusters without detectable sequence similarity, raising the question of whether they were homologous or instead had originated independently. Since TupV C and SeV C are representative of these two clusters, our discovery that they have a similar structure indi-cates that all Orthoparamyxovirine C proteins are homologous. Our results also imply that, strik-ingly, a STAT1-binding site is encoded by exactly the same RNA region of the P/C gene across Paramyxovirinae, but in different reading frames (P or C) depending on which cluster they belong to.