Hepatitis C virus (HCV), an important pathogen and prototype hepacivirus, 5’ caps its RNA with flavin adenine dinucleotide (FAD); no other 5’ metabolite capped viral RNAs have been identified. Here, we establish NADzymeID, allowing specific identification of 5’ nicotinamide adenine dinucleotide (NAD). Using NADzymeID, we find that Norway rat hepacivirus 1 (NrHV-1) and related hepaciviruses use 5’ NAD capping, whereas the closest relatives of HCV use 5’ FAD, thereby revealing evolutionary divergence of 5’ capping. NrHV-1 replication is attenuated in absence of NAD, and NrHV-1 is cleared more rapidly in mice with reduced intrahepatic NAD levels. As observed for 5’ FAD, 5’ NAD protects viral RNA from RIG-I mediated innate immune sensing. Thus, despite evolutionary divergence, metabolite capping generally confers evasion of innate immune responses. These represent the first examples of 5’ NAD capping for viruses, providing tools also to investigate the elusive function of cellular 5’ metab

Hepacivirus bovis (BovHepV) has been detected globally since its initial description in 2015, but its transmission routes remain largely unknown. Arthropod-borne or vertical transmission is a common route for several livestock viruses, including Rift Valley fever virus and bluetongue virus. Moreover, many members of the genus Orthoflavivirus (family Flaviviridae) are transmitted by hematophagous arthropods, raising the possibility that hepaciviruses such as BovHepV, which also belongs to the...

Rodents are substantial reservoirs of zoonotic viruses with regular human exposure restricted to a limited number of species. Numerous rodent species have been shown to harbor emerging viruses, inc...

What happens if you investigate 986 ticks, representing six species, collected from the diverse ecological landscapes of Hebei Province in n...

by Yasunori Akaike, Tomohisa Tanaka, Hirotake Kasai, Atsuya Yamashita, Yoshiharu Matsuura, Kohji Moriishi The lack of immuno-competent animal models of hepatitis C virus (HCV) infection has been an obstacle to vaccine development and research on immune responses. Hepacivirus ratti (Norway rat hepacivirus-1: NRHV1) is a virus closely related to HCV that specifically infects the liver and induces hepatocellular carcinoma in rats, making it a promising surrogate model for HCV. NRHV1 expressing a reporter gene serves as a powerful tool for analyzing the in vivo dynamics and pathogenicity mechanisms of NRHV1. In this study, we developed a reporter NRHV1 capable of infection and replication in both immunodeficient mice and cultured cells and established a platform for generating diverse reporter viruses. A reporter virus containing the HiBiT gene in the coding region of NS5A domain III was constructed using circular polymerase extension reaction (CPER). Infection with this reporter virus led to HiBiT activity in infected cells and the activity was correlated with the amount of intracellular viral RNA. In addition, this reporter virus established persistent infection in NOD-SCID mice and led to the generation of HiBiT activity in the livers of infected mice, although loss of the HiBiT gene was observed in some mice. Furthermore, reporter virus recovered from infected mice could infect and generate HiBiT activity in cultured cells. These results demonstrate that the reporter virus can infect hepatocytes in vivo and in vitro. This platform provides a versatile tool for in vitro quantitative antiviral screening and for exploring viral infection dynamics in vivo, although further studies will be required to improve the long-term stability of the HiBiT reporter.

by Yasunori Akaike, Tomohisa Tanaka, Hirotake Kasai, Atsuya Yamashita, Yoshiharu Matsuura, Kohji Moriishi The lack of immuno-competent animal models of hepatitis C virus (HCV) infection has been an obstacle to vaccine development and research on immune responses. Hepacivirus ratti (Norway rat hepacivirus-1: NRHV1) is a virus closely related to HCV that specifically infects the liver and induces hepatocellular carcinoma in rats, making it a promising surrogate model for HCV. NRHV1 expressing a reporter gene serves as a powerful tool for analyzing the in vivo dynamics and pathogenicity mechanisms of NRHV1. In this study, we developed a reporter NRHV1 capable of infection and replication in both immunodeficient mice and cultured cells and established a platform for generating diverse reporter viruses. A reporter virus containing the HiBiT gene in the coding region of NS5A domain III was constructed using circular polymerase extension reaction (CPER). Infection with this reporter virus led to HiBiT activity in infected cells and the activity was correlated with the amount of intracellular viral RNA. In addition, this reporter virus established persistent infection in NOD-SCID mice and led to the generation of HiBiT activity in the livers of infected mice, although loss of the HiBiT gene was observed in some mice. Furthermore, reporter virus recovered from infected mice could infect and generate HiBiT activity in cultured cells. These results demonstrate that the reporter virus can infect hepatocytes in vivo and in vitro. This platform provides a versatile tool for in vitro quantitative antiviral screening and for exploring viral infection dynamics in vivo, although further studies will be required to improve the long-term stability of the HiBiT reporter.

The NS3 helicases from the Flaviviridae family of viruses exhibit nucleotide-hydrolysis-dependent, nucleic-acid-unwinding activity. The RNA unwinding activity for NS3 helicases from the Orthoflavivirus genus has not been fully explored and contrasts with NS3 helicase from Hepatitis C virus (HCV) of the Hepacivirus genus, which has thus far served as the prototypical model enzyme from this family of viruses. To begin to understand the functional differences between flavivirus NS3 helicases, we first developed an expression and purification system for full-length untagged NS3 protein from West Nile virus (WNV) and Zika virus (ZIKV). Both enzymes exhibit RNA-stimulated ATPase activity and are dependent on the nucleoside triphosphatase active site of the enzyme. Unlike HCV NS3, orthoflavivirus NS3s do not efficiently pre-assemble on a 3-ssRNA-tailed dsRNA substrate in the absence of ATP-Mg which is a prerequisite for formation of a productive HCV NS3-RNA complex that can exhibit a rapid burst of RNA unwinding. Instead, to observe RNA unwinding by WNV and ZIKV NS3s, low Mg-ATP concentrations are required at a time coincident when NS3 encounters the RNA substrate. In addition, we find that orthoflavivirus NS3s require translocation beyond the displaced strand to completely unwind a dsRNA substrate. Last, we find that orthoflavivirus NS5 stimulates the ability of NS3 to unwind dsRNA. These results suggest that functional differences exist between the flavivirus NS3 helicases and illuminate that orthoflavivirus NS3s require a functional interaction with the NS5 protein for coordination of its activity, as it is believed these two proteins constitute the viral replicase.

The NS3 helicases from the Flaviviridae family of viruses exhibit nucleotide-hydrolysis-dependent, nucleic-acid-unwinding activity. The RNA unwinding activity for NS3 helicases from the Orthoflavivirus genus has not been fully explored and contrasts with NS3 helicase from Hepatitis C virus (HCV) of the Hepacivirus genus, which has thus far served as the prototypical model enzyme from this family of viruses. To begin to understand the functional differences between flavivirus NS3 helicases, we first developed an expression and purification system for full-length untagged NS3 protein from West Nile virus (WNV) and Zika virus (ZIKV). Both enzymes exhibit RNA-stimulated ATPase activity and are dependent on the nucleoside triphosphatase active site of the enzyme. Unlike HCV NS3, orthoflavivirus NS3s do not efficiently pre-assemble on a 3-ssRNA-tailed dsRNA substrate in the absence of ATP-Mg which is a prerequisite for formation of a productive HCV NS3-RNA complex that can exhibit a rapid burst of RNA unwinding. Instead, to observe RNA unwinding by WNV and ZIKV NS3s, low Mg-ATP concentrations are required at a time coincident when NS3 encounters the RNA substrate. In addition, we find that orthoflavivirus NS3s require translocation beyond the displaced strand to completely unwind a dsRNA substrate. Last, we find that orthoflavivirus NS5 stimulates the ability of NS3 to unwind dsRNA. These results suggest that functional differences exist between the flavivirus NS3 helicases and illuminate that orthoflavivirus NS3s require a functional interaction with the NS5 protein for coordination of its activity, as it is believed these two proteins constitute the viral replicase.

We talk with Dr. John Gridley, a postdoctoral fellow in the Vaccinology Training Program at Emory University, GA, who uses a hepatitis C virus-related rodent hepacivirus model to identify correlates o...

Author summary Vaccine development for hepatitis C virus (HCV) is currently impeded by the lack of suitable animal models. To address this, the Norway rat hepacivirus 1 (NrHV), a virus relatively clos...