Abstract:To monitor the arthropod-borne virus transmission in mosquitoes, we have attempted both to detect and isolate viruses from 3304 wild-caught female mosquitoes in the Livingstone (Southern Province) and Mongu (Western Province) regions in Zambia in 2017. A pan-flavivirus RT-PCR assay was performed to identify flavivirus genomes in total RNA extracted from mosquito lysates, followed by virus isolation and full genome sequence analysis using next-generation sequencing and rapid amplification of cDNA ends. We isola… Show more
“…The MPFV DB element is of particular interest, given that DB elements were typically not found in the 3′-UTRs of mammalian or seabird-associated TBFVs, but were present in several TBFV-related NKVs 30 , with which MPFV shared ancestral roots. Although the proximal stem-loop originating from the central multi-loop of the MPFV DB element was considerably shorter than the DB elements of MBFVs or dual-host affiliated ISFVs 30 , 45 , for example, MPFV was unique among the known TBFVs to contain such an element in its 3′-UTR. Figure 5 Predicted secondary structures of the 5′- and the 3′-UTRs of MPFV.…”
Tick-borne flaviviruses (TBFVs) infect mammalian hosts through tick bites and can cause various serious illnesses, such as encephalitis and hemorrhagic fevers, both in humans and animals. Despite their importance to public health, there is limited epidemiological information on TBFV infection in Africa. Herein, we report that a novel flavivirus, Mpulungu flavivirus (MPFV), was discovered in a Rhipicephalus muhsamae tick in Zambia. MPFV was found to be genetically related to Ngoye virus detected in ticks in Senegal, and these viruses formed a unique lineage in the genus Flavivirus. Analyses of dinucleotide contents of flaviviruses indicated that MPFV was similar to those of other TBFVs with a typical vertebrate genome signature, suggesting that MPFV may infect vertebrate hosts. Bioinformatic analyses of the secondary structures in the 3′-untranslated regions (UTRs) revealed that MPFV exhibited unique exoribonuclease-resistant RNA (xrRNA) structures. Utilizing biochemical approaches, we clarified that two xrRNA structures of MPFV in the 3′-UTR could prevent exoribonuclease activity. In summary, our findings provide new information regarding the geographical distribution of TBFV and xrRNA structures in the 3′-UTR of flaviviruses.
“…The MPFV DB element is of particular interest, given that DB elements were typically not found in the 3′-UTRs of mammalian or seabird-associated TBFVs, but were present in several TBFV-related NKVs 30 , with which MPFV shared ancestral roots. Although the proximal stem-loop originating from the central multi-loop of the MPFV DB element was considerably shorter than the DB elements of MBFVs or dual-host affiliated ISFVs 30 , 45 , for example, MPFV was unique among the known TBFVs to contain such an element in its 3′-UTR. Figure 5 Predicted secondary structures of the 5′- and the 3′-UTRs of MPFV.…”
Tick-borne flaviviruses (TBFVs) infect mammalian hosts through tick bites and can cause various serious illnesses, such as encephalitis and hemorrhagic fevers, both in humans and animals. Despite their importance to public health, there is limited epidemiological information on TBFV infection in Africa. Herein, we report that a novel flavivirus, Mpulungu flavivirus (MPFV), was discovered in a Rhipicephalus muhsamae tick in Zambia. MPFV was found to be genetically related to Ngoye virus detected in ticks in Senegal, and these viruses formed a unique lineage in the genus Flavivirus. Analyses of dinucleotide contents of flaviviruses indicated that MPFV was similar to those of other TBFVs with a typical vertebrate genome signature, suggesting that MPFV may infect vertebrate hosts. Bioinformatic analyses of the secondary structures in the 3′-untranslated regions (UTRs) revealed that MPFV exhibited unique exoribonuclease-resistant RNA (xrRNA) structures. Utilizing biochemical approaches, we clarified that two xrRNA structures of MPFV in the 3′-UTR could prevent exoribonuclease activity. In summary, our findings provide new information regarding the geographical distribution of TBFV and xrRNA structures in the 3′-UTR of flaviviruses.
“…Since Xrn-1 has been suggested to be involved in the degradation of damaged RNA, ( Boo and Kim, 2020 ) the results that display enzyme stalling in the presence of 8-oxoG were surprising, therefore we considered structural parameters as a possible explanation. Although the canonical strand 1 ″ resulted in a suitable Xrn-1 substrate, with efficient degradation, we considered two aspects that led us to probe for RNA structure; 1) that Xrn-1 induced degradation of RNA is halted upon encountering highly structured RNA (albeit much more structured than the 20-mers studied in this work); ( Wastika et al, 2020 ) and 2) that the presence of one 8-oxoG lesion can result in structural changes even when using short oligonucleotides of RNA ( Choi et al, 2017 ). A combination of CD with an established web server ( Zuker, 2003 ) allowed us to establish the formation of secondary structure and predict the potential folds of ONs 1 – 3 and 6 – 8 .…”
Understanding how oxidatively damaged RNA is handled intracellularly is of relevance due to the link between oxidized RNA and the progression/development of some diseases as well as aging. Among the ribonucleases responsible for the decay of modified (chemically or naturally) RNA is the exonuclease Xrn-1, a processive enzyme that catalyzes the hydrolysis of 5′-phosphorylated RNA in a 5′→3′ direction. We set out to explore the reactivity of this exonuclease towards oligonucleotides (ONs, 20-nt to 30-nt long) of RNA containing 8-oxo-7,8-dihydroguanosine (8-oxoG), obtained via solid-phase synthesis. The results show that Xrn-1 stalled at sites containing 8-oxoG, evidenced by the presence of a slower moving band (via electrophoretic analyses) than that observed for the canonical analogue. The observed fragment(s) were characterized via PAGE and MALDI-TOF to confirm that the oligonucleotide fragment(s) contained a 5′-phosphorylated 8-oxoG. Furthermore, the yields for this stalling varied from app. 5–30% with 8-oxoG located at different positions and in different sequences. To gain a better understanding of the decreased nuclease efficiency, we probed: 1) H-bonding and spatial constraints; 2) anti-syn conformational changes; 3) concentration of divalent cation; and 4) secondary structure. This was carried out by introducing methylated or brominated purines (m1G, m6,6A, or 8-BrG), probing varying [Mg2+], and using circular dichroism (CD) to explore the formation of structured RNA. It was determined that spatial constraints imposed by conformational changes around the glycosidic bond may be partially responsible for stalling, however, the results do not fully explain some of the observed higher stalling yields. We hypothesize that altered π-π stacking along with induced H-bonding interactions between 8-oxoG and residues within the binding site may also play a role in the decreased Xrn-1 efficiency. Overall, these observations suggest that other factors, yet to be discovered/established, are likely to contribute to the decay of oxidized RNA. In addition, Xrn-1 degraded RNA containing m1G, and stalled mildly at sites where it encountered m6,6A, or 8-BrG, which is of particular interest given that the former two are naturally occurring modifications.
“…So far, such truncated sfRNAs have only been observed in YFV [ 100 ]. Two sfRNAs have been reported in TBEV ( Figure 4 b), and a single sfRNA has been detected for the ISFV and NKFV group members ( Figure 4 c,d) [ 101 , 102 , 103 ].…”
The genus Flavivirus comprises numerous, small, single positive-stranded RNA viruses, many of which are important human pathogens. To store all the information required for their successful propagation, flaviviruses use discrete structural genomic RNA elements to code for functional information by the establishment of dynamic networks of long-range RNA–RNA interactions that promote specific folding. These structural elements behave as true cis-acting, non-coding RNAs (ncRNAs) and have essential regulatory roles in the viral cycle. These include the control of the formation of subgenomic RNAs, known as sfRNAs, via the prevention of the complete degradation of the RNA genome. These sfRNAs are important in ensuring viral fitness. This work summarizes our current knowledge of the functions performed by the genome conformations and the role of RNA–RNA interactions in these functions. It also reviews the role of RNA structure in the production of sfRNAs across the genus Flavivirus, and their existence in related viruses.
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