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)

Reduced Nucleoprotein Availability Impairs Negative-Sense RNA Virus Replication and Promotes Host Recognition

Nucleoprotein is important in regulating transcription and replication of Negative-sense RNA viruses. Although the viral RNA-dependent RNA-polymerase can induce the synthesis of viral RNA in the absence of nucleoprotein, its processivity is severely impaired. During the early stages of infection, nucleoprotein supports transcription of viral proteins, which thereafter package and protect replication products. This study shows that limited levels of nucleoprotein in Influenza A and Sendai viruses result in impaired replication and formation of defective viral genomes which are detected by the host, hence inducing a robust Interferon response. Seven different viruses (with limited nucleoprotein levels) belonging to five families in the phylum Negarnaviricota infected a significantly low number of cells, except for two viruses, further confirming the importance of nucleoprotein in the replication of these viruses. Reduced levels of nucleocapsid expression in SARS-CoV-2 resulted in reduced replication, with no induction of interferon response, due to the non-inflammatory nature of positive-sense viral genomes. This highlights the importance of nucleoprotein and nucleocapsid in efficient viral replication while playing a critical role of protecting the virus from recognition by the host’s defense mechanism. These insights provide a new rationale for developing anti-viral strategies targeting the nucleoprotein. 

(MRM)

Two amino acid pairs in the glycoprotein of severe fever with thrombocytopenia syndrome virus responsible for the enhanced virulence

Severe fever with thrombocytopenia syndrome (SFTS) virus poses a major public health threat, with high mortality rates in both humans and cats. This study investigates the genetic factors underlying the differing virulence of SFTS virus (SFTSV) strains Tk-F123 and Ng-F264, isolated from cats. In type I interferon receptor-knockout mice, the Tk-F123 strain was uniformly lethal, while the Ng-F264 strain caused no fatalities. A critical difference was identified in the Gc protein: Tk-F123 encodes glycine and serine at residues 581 and 934, respectively, whereas Ng-F264 encodes arginine and asparagine at these positions. These amino acid differences significantly impact Gc protein function, modulating SFTSV virulence. These findings offer valuable insights into the molecular mechanisms of SFTSV pathogenicity and could guide the development of live-attenuated vaccines and antiviral therapies.
(SN)

Hsp90 β is critical for the infection of severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome (SFTS) caused by the SFTS virus (SFTSV) is an emerging disease in East Asia with a fatality rate of up to 30%. SFTS is often characterized by an acute fever accompanied by thrombocytopenia and leukocytopenia, which can cause bleeding and severe consequences. The SFTSV genome is composed of three segments of single-stranded, negative-sense RNA, including large (L), middle (M), and small (S) segments. The S segment applies an ambisense strategy to encode nucleoprotein (NP) and nonstructural protein (NSs). NSs is a virulence factor of SFTSV. This study investigated the role of heat-shock protein 90 (Hsp90) during SFTSV infection. The association between NSs and Hsp90 isoforms was also explored. The data showed Hsp90 inhibitors could significantly inhibit the SFTSV infection markedly decreased the expression of the SFTSV encoded main virulence factor, NSs, in SFTSV infected cells. This study provides new insight into the regulation of NSs expression by host factors and suggests that Hsp90 inhibitors may exert therapeutic benefits for SFTS disease.
(DKW)

Identification of Host Factors for Rift Valley Fever Phlebovirus

A zoonotic pathogen Rift Valley Fever phlebovirus is well circulated among humans and livestock, characterized as causing abortion with almost 100% mortality rates in newborn animals. Presently, lack of FDA-approved antiviral drug/ license vaccine to control RFV in humans and detailed knowledge of viral protein function is necessary to successfully develop antiviral therapies. RVFV is susceptible to infect and replicate in a variety of cell types from frogs, pigs, elk, mule deer, reptiles. To analyze the host factor role, CRISPR-Cas9 knockout screening in A549 human cells and validated the function of the selected gene candidates by measuring the intracellular viral RNA accumulation, western blot, and RT-qPCR for two viruses, RVFV and La Crosse Encephalitis Virus (LACV). A gene knockout of member of WD repeat protein family (WDR7) in A549 human cell line is confirmed as important factor in the replication of two different viruses from Bunyavirales order. WDR7 gene disruption affects intracellular viral RNA accumulation primarily in the late phase of RVFV replication cycle and at the early phase of the LACV replication cycle. Moreover, the finding also highlight that WDR7 could be used as potential drug target for further antiviral development.
(INV)