Subgenomic flavivirus RNA (sfRNA) associated with Asian lineage Zika virus identified in three species of Ugandan bats (family Pteropodidae)

Anna C Fagre,Betty Nalikka,Juliette Lewis,Luke Nyakarahuka,Brian Foy,Teddy Nakayiki,Eric C Mossel,Tony Schountz,Robert Kityo,Rebekah C Kading,Jonathan S Towner,Julius J Lutwama,Brian R Amman,John Anderson,Megan R Miller,Tara K Sealy

doi:10.1038/s41598-021-87816-5

Abstract

Serological cross-reactivity among flaviviruses makes determining the prior arbovirus exposure of animals challenging in areas where multiple flavivirus strains are circulating. We hypothesized that prior infection with ZIKV could be confirmed through the presence of subgenomic flavivirus RNA (sfRNA) of the 3′ untranslated region (UTR), which persists in tissues due to XRN-1 stalling during RNA decay. We amplified ZIKV sfRNA but not NS5 from three experimentally-infected Jamaican fruit bats, supporting the hypothesis of sfRNA tissue persistence. Applying this approach to 198 field samples from Uganda, we confirmed presence of ZIKV sfRNA, but not NS5, in four bats representing three species: Eidolon helvum (n = 2), Epomophorus labiatus (n = 1), and Rousettus aegyptiacus (n = 1). Amplified sequence was most closely related to Asian lineage ZIKV. Our results support the use of sfRNA as a means of identifying previous flavivirus infection and describe the first detection of ZIKV RNA in East African bats.

Highlights

The MR766 strain of ZIKV was originally isolated from a sentinel rhesus macaque (Macaca mulatta) in the canopy of Zika forest in 1 9472, where bats reside
Challenged bats were screened for ZIKV subgenomic flaviviral RNA (sfRNA) using the droplet digital PCR
Successful amplification and Sanger sequencing of ZIKV 3′untranslated region (UTR) sfRNA, but not NS5, from multiple organs and blood from Jamaican fruit bats subcutaneously inoculated with ZIKV (Table 2) further supports our hypothesis that sfRNA is a more sensitive detection target than NS5 due to XRN1 stalling

Summary

Introduction

The MR766 strain of ZIKV was originally isolated from a sentinel rhesus macaque (Macaca mulatta) in the canopy of Zika forest in 1 9472, where bats reside. RNA viruses have evolved mechanisms to combat degradation by these enzymes and protect viral nucleic acid for replication and other purpose[22] Among these mechanisms, subgenomic flaviviral RNA (sfRNA) in the 3′ untranslated region (UTR) is known to persist at higher levels in host tissue than genomic RNA, due to its ability to stall exoribonuclease-1 (XRN-1) on the complex hairpin structures characteristic of viral 3′ sequences. Subgenomic flaviviral RNA (sfRNA) in the 3′ untranslated region (UTR) is known to persist at higher levels in host tissue than genomic RNA, due to its ability to stall exoribonuclease-1 (XRN-1) on the complex hairpin structures characteristic of viral 3′ sequences This stalling results in incomplete degradation of viral transcripts and subsequent accumulation of these short, subgenomic sequences (sfRNA) in cells and tissue[23,24,25]. It describes the application of sfRNA as a target for detection of residual viral RNA in free-ranging wildlife

Methods

Results

Conclusion