Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof

Reverse genetics is a crucial tool for studying virus replication and pathogenesis in negative strand RNA viruses (NSVs). However, its application has been limited by a scarcity of infectious cDNA clones from virulent field strains, as most available clones originate from cell-adapted or attenuated viruses, hindering pathogenesis research. To overcome this, a novel high-throughput system was developed for directly cloning complete NSV genomes into reverse genetics vectors via linear-to-linear RedE/T recombination (LLHR). This method demonstrated reliability by rapidly cloning multiple rabies virus (RABV) full-length genomes, achieving 73% to 88% insertion rates identical to field virus consensus. Recombinant RABVs were successfully recovered, exhibiting comparable growth kinetics, preserved cell type-specific replication, and virulence in mouse models. The system’s flexibility was also confirmed by efficiently cloning an orthobunyavirus L genome segment. This technological advance significantly improves the analysis of virus variability and phenotypical characterization of recombinant viruses at a clonal level boosting future pathogenesis research.
(MKO)

TRIM44 Promotes Rabies Virus Replication by Autophagy-Dependent Mechanism

The tripartite motif (TRIM) proteins control essential biological processes, and have been shown to play important roles in viral infections. This study showed that Rabies virus (RABV) infection to neuroblastoma cells caused upregulation of TRIM47, TRIM56 and TRIM44. Although silencing of the former two did not have any significant effect on the transcription of the RABV viral genome, silencing of TRIM44 significantly inhibited the RABV genome and the virus titre. Furthermore, knockdown of TRIM44 reduced the viral titre and the expression of the glycoprotein (G), while its overexpression led to increased transcription of the RABV viral genome and the G protein. Depletion of TRIM44 in neuroblastoma cells led to a reduced expression of the RABV nucleoprotein. The addition of an autophagy inhibitor reversed the potentiating effect of TRIM44 on G protein expression and genome replication, while the addition of an autophagy inducer enhanced the TRIM44-induced expression of the RABV glycoprotein. This study highlighted the crucial role of TRIM44 in RABV replication. Further research is needed to identify other mechanisms by which TRIM44 regulates RABV infection.
(MRM)

A positive-sense single-stranded RNA virus acquired a negative-sense open reading frame through recombination

Single stranded RNA viruses are broadly classified into positive-sense and negative-sense single-stranded RNA. This distinction is due to the different transcription and replication mechanisms. In this study, researchers identified and analyzed an RNA virus, Brine Shrimp Virga-like Virus 1 (BSVV1) found in brine shrimp. BSVV1’s genome included three Open Reading Frames (ORFs). ORF1 resembled the RdRp of Ips virga-like virus 1. ORF2 showed links to Hubei bunya-like virus 10. ORF3 had no known homologs. ORF2 was transcribed directly as in positive-sense viruses, but closely resembled the glycoprotein of various negative-sense bunyaviruses. This suggested ancient recombination between positive-sense and negative-sense RNA viruses. TEM imaging revealed non-enveloped viral particles (~50nm), but further studies are essential to confirm envelope absence or presence. The study established BSVV1 is globally distributed although its ecological spread is unclear requiring more research. BSVV1’s novel genome sequence shows significant evidence of recombination between distinct RNA virus phyla, offering new insights into virus evolution and diversity. Further studies should focus on understanding BSVV1’s protein functions and its impact on viral adaption.
(MKO)