The polymerase of negative stranded RNA viruses

Negative-sense RNA viruses are divided into non segmented viruses such as Ebola and segmented viruses such as influenza. Both groups protect their genomic RNA inside a nucleocapsid protein(NP) that the viral RNA-Dependent RNA polymerase must navigate during transcription and replication. Both classes share similar polymerase core architecture but their capping and structural mechanisms are different. NNS viruses use internal PRNTase and MTase domains to synthesize fully modified 5’ caps and have rigid locked nucleoplasmids. SNS viruses have loosely organized nucleocaspids and hijack host mRNA caps via a cap dependent endonuclease domain. In NNs systems the phosphoprotein (P) is an important cofactor that changes the L proteins structural appendage and acts as a processivity factor whereas SNS polymerases  accesses templates directly. Recent Invitro assays have shown that polymerases can initiate denovo or using primers on naked RNA through precise 3’ terminal base pairing. Also template associated protein NP remains important to achieve full processivity. 
(MCW)

Development of a strand-specific RT-qPCR assay for detecting and quantifying Borna disease virus RNAs in infected cells

BoDV-1 (Borna disease virus 1) is a negative-sense RNA virus that establishes persistent infection in the mammalian central nervous system and has recently been associated with fatal encephalitis in humans. During replication, BoDV-1 produces three types of RNA: genomic RNA, antigenomic RNA, and mRNA. However, conventional RT-qPCR cannot distinguish these RNA species. This study aimed to develop a strand-specific RT-qPCR assay capable of detecting and quantifying each viral RNA individually. Standard RNAs were prepared using the 5′- and 3′-terminal 1,200-nucleotide regions of the viral genome, the full-length N mRNA, and the 3′ region of L mRNA. Strand specificity was achieved by using tagged RT primers and increasing the reverse transcription temperature to 57°C. Vero cells infected with BoDV-1 were monitored for one month. The assay specifically detected genomic and antigenomic RNAs, whereas N mRNA could not be completely distinguished from antigenomic RNA because of their highly similar sequences. Therefore, N mRNA levels were estimated by subtracting the antigenomic RNA signal. Viral RNA levels increased during infection, while the antigenome-to-genome ratio remained constant, suggesting stable viral replication. In contrast, the mRNA-to-genome ratio gradually decreased, indicating transcriptional suppression during persistent infection. Limitations of the study include the use of synthetic standard RNAs, experiments performed only in Vero cells, and the analysis of only N mRNA among the viral transcripts.
(AI)

The development of biologically contained Sudan virus as an alternative and safe tool for fundamental filovirus research

Filoviruses, including Ebola virus, Sudan virus, and Marburg virus, cause severe diseases with high fatality rates and require BSL-4 containment, which significantly restricts research. Although several BSL-2 surrogate systems have been developed, they often fail to fully reproduce viral replication and the complete life cycle. VP30 is a transcriptional activator essential for initiating viral mRNA synthesis in filoviruses and is functionally exchangeable among related viruses. VP30-deficient filovirus systems have been reported; however, a VP30-deficient Sudan virus (SUDV) system has not previously been established. In the reported study, a SUDV genome in which the VP30 gene was replaced with GFP was transfected into Vero cells expressing SUDV VP30. GFP fluorescence confirmed successful virus rescue. The rescued virus was passaged, and a VP30-deficient SUDV was isolated. Sequencing confirmed no unintended mutations, and no VP30 reversion was observed after 10 passages. Growth analysis showed replication only in VP30-expressing cells, while no replication occurred in VP30-negative cells. VP30 proteins from EBOV, RESTV, and SUDV showed high functional compatibility, whereas MARV VP30 showed little complementation. This system provides a platform that may reduce reliance on BSL-4 containment and facilitate safer studies of SUDV biology, antiviral development, and filovirus evolution.
(AI)