Recent Advances in Bunyavirus Reverse Genetics Research: Systems Development, Applications, and Future Perspectives

Abstract

Bunyaviruses are members of the Bunyavirales order, which is the largest group of RNA viruses, comprising 12 families, including a large group of emerging and re-emerging viruses. These viruses can infect a wide variety of species worldwide, such as arthropods, protozoans, plants, animals, and humans, and pose substantial threats to the public. In view of the fact that a better understanding of the life cycle of a highly pathogenic virus is often a precondition for developing vaccines and antivirals, it is urgent to develop powerful tools to unravel the molecular basis of the pathogenesis. However, biosafety level −3 or even −4 containment laboratory is considered as a necessary condition for working with a number of bunyaviruses, which has hampered various studies. Reverse genetics systems, including minigenome (MG), infectious virus-like particle (iVLP), and infectious full-length clone (IFLC) systems, are capable of recapitulating some or all steps of the viral replication cycle; among these, the MG and iVLP systems have been very convenient and effective tools, allowing researchers to manipulate the genome segments of pathogenic viruses at lower biocontainment to investigate the viral genome transcription, replication, virus entry, and budding. The IFLC system is generally developed based on the MG or iVLP systems, which have facilitated the generation of recombinant infectious viruses. The MG, iVLP, and IFLC systems have been successfully developed for some important bunyaviruses and have been widely employed as powerful tools to investigate the viral replication cycle, virus–host interactions, virus pathogenesis, and virus evolutionary process. The majority of bunyaviruses is generally enveloped negative-strand RNA viruses with two to six genome segments, of which the viruses with bipartite and tripartite genome segments have mostly been characterized. This review aimed to summarize current knowledge on reverse genetic studies of representative bunyaviruses causing severe diseases in humans and animals, which will contribute to the better understanding of the bunyavirus replication cycle and provide some hints for developing designed antivirals.

SFTS bunyavirus NSs protein sequestrates mTOR into inclusion bodies and deregulates mTOR‐ULK1 signaling, provoking pro‐viral autophagy

“…Thus, we next tested the potential effects of autophagy on the core machinery of SFTSV replication, RNP, by exploiting a reverse genetic minigenome system that is generally used as the reporter of RNP activity. As previously described, the minigenome system consists of NP and RdRp expression plasmids, along with a minigenome reporter plasmid encoding a viral genome analog containing firefly luciferase sequence flanked by M segment untranslated regions (UTRs) 35 . As shown in Figure 7E, NP and RdRp significantly triggered minigenome system activity compared with the corresponding control plasmids (vector), indicating that functional RNPs are generated and functioning.…”

“…As previously described, the minigenome system consists of NP and RdRp expression plasmids, along with a minigenome reporter plasmid encoding a viral genome analog containing firefly luciferase sequence flanked by M segment untranslated regions (UTRs). 35 As shown in Figure 7E, NP and RdRp significantly triggered minigenome system activity compared with the corresponding control plasmids (vector), indicating that functional RNPs are generated and functioning. Interestingly, treatment with autophagy inducer rapamycin further enhanced the minigenome system activity in wildtype cells; however, in Beclin1-KO cells, the reporter activity was significantly decreased compared with that in the control cells, suggesting that autophagy likely promotes the replication/ transcription machinery RNP of SFTSV.…”

“…The SFTSV minigenome system consists of expression plasmids for NP and RdRp proteins and a reporter plasmid transcribing reporter gene sequence fused with M genomic UTRs (called pMUTR-Luc), as described previously. 35 The wild-type and Beclin-1 KO cells plated on 24-well plates were transfected with 300 ng/well pMUTR-Luc reporter plasmid, 10 ng/well pRL-TK internal reference plasmid, 300 ng/well RdRp expression plasmid and 100 ng/well NP expression plasmid or the corresponding empty plasmid. At 12 h posttransfection, cells were treated with indicated drugs or DMSO for 24 h, and then the activities of luciferase were measured with a dual luciferase reporter kit (Promega, Cat#E2940).…”

Mapping the viruses belonging to the order Bunyavirales in China

“…The Bunyavirales is a large RNA virus order and comprises more than 560 viruses divided into twelve families [1], among which most members could infect humans and animals, thus posing a great potential threat to public health [2]. For example, hantaviruses usually cause two clinical syndromes when infecting humans, including hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS).…”

A comprehensive list of the replication promoters of Bunyavirales reveals a unique promoter structure in Nairoviridae differing from other virus families

“…It consists of 12 families of closely related viruses: Arenaviridae, Cruliviridae, Fimoviridae, Hantaviridae, Leishbuviridae, Mypoviridae, Nairoviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae, Tospoviridae , and Wupedeviridae . Members of Bunyavirales have a segmented single-stranded negative-sense or ambisense RNA genome (1,2). The families Arenaviridae, Hantaviridae, Nairoviridae, Peribunyaviridae , and Phenuiviridae include several important pathogens that can cause severe diseases in animals, including humans, while the families Fimoviridae, Phasmaviridae, Phenuiviridae , and Tospoviridae include pathogens associated with plant diseases.…”