Development of a semicomprehensive detection method for paramyxoviruses and its validation using Indonesian bats.

An outbreak of zoonotic diseases is one of the worldwide threats. Bats were reported as important reservoir hosts for many emerging zoonotic diseases. To mitigate the risk, understanding bat virome and their distribution is indispensable. Universal detection methods that can simultaneously identify multiple viruses are some of the most promising approaches. Here, we developed a semicomprehensive detection method integrating group-wide RT-PCR for paramyxoviruses and multiplex next-generation sequencing. The RT-PCR consists of three sets of degenerative primers covering viruses from Paramyxoviridae, including Pneumoviridae, which have now been reclassified into a distinct family. Index nucleotides were added to the primers to enable cost-effective multiplex sequencing, and the length of index was optimized to increase sensitivity. The method was applied to tracheal and rectal swabs from 135 bats captured in Indonesia. A conventional RT-PCR test validated the NGS results. Collectively, seven sequences of novel paramyxovirus-like similar to Pararubulavirus, Orthorubulavirus, and Henipavirus were successfully identified from seven bat samples. Furthermore, sequences between the two different target locations detected by NGS in the virus genomes were verified by RT-PCR. The similarity of the obtained sequences to the known paramyxoviruses sequences was relatively low, ranging from 70.88 to 82.44%. It suggests that the obtained sequences from novel viruses and the zoonotic risk of those novel viruses remain unknown. This cost-affordable, semi-comprehensive, pan-paramyxovirus test can be applied to other samples for viral genome surveillance, and the same strategy can be implemented to other pathogens for zoonosis control.