A novel nairovirus associated with acute febrile illness in Hokkaido, Japan.

The increasing burden of tick-borne orthonairovirus infections, such as Crimean-Congo hemorrhagic fever, is becoming a global concern for public health. In the present study, we identify a novel orthonairovirus, designated Yezo virus (YEZV), from two patients showing acute febrile illness with thrombocytopenia and leukopenia after tick bite in Hokkaido, Japan, in 2019 and 2020, respectively. YEZV is phylogenetically grouped with Sulina virus detected in Ixodes ricinus ticks in Romania. YEZV infection has been confirmed in seven patients from 2014-2020, four of whom were co-infected with Borrelia spp. Antibodies to YEZV are found in wild deer and raccoons, and YEZV RNAs have been detected in ticks from Hokkaido. In this work, we demonstrate that YEZV is highly likely to be the causative pathogen of febrile illness, representing the first report of an endemic infection associated with an orthonairovirus potentially transmitted by ticks in Japan.

X-ray inactivation of RNA viruses without loss of biological characteristics.

In the event of an unpredictable viral outbreak requiring high/maximum biosafety containment facilities (i.e. BSL3 and BSL4), X-ray irradiation has the potential to relieve pressures on conventional diagnostic bottlenecks and expediate work at lower containment. Guided by Monte Carlo modelling and in vitro 1-log10 decimal-reduction value (D-value) predictions, the X-ray photon energies required for the effective inactivation of zoonotic viruses belonging to the medically important families of Flaviviridae, Nairoviridae, Phenuiviridae and Togaviridae are demonstrated. Specifically, it is shown that an optimized irradiation approach is attractive for use in a multitude of downstream detection and functional assays, as it preserves key biochemical and immunological properties. This study provides evidence that X-ray irradiation can support emergency preparedness, outbreak response and front-line diagnostics in a safe, reproducible and scalable manner pertinent to operations that are otherwise restricted to higher containment BSL3 or BSL4 laboratories.

Rescue of Infectious Recombinant Hazara Nairovirus from cDNA Reveals the Nucleocapsid Protein DQVD Caspase Cleavage Motif Performs an Essential Role other than Cleavage.

The Nairoviridae family of the Bunyavirales order comprises tick-borne, trisegmented, negative-strand RNA viruses, with several members being associated with serious or fatal diseases in humans and animals. A notable member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is the most widely distributed tick-borne pathogen and is associated with devastating human disease, with case fatality rates averaging 30%. Hazara virus (HAZV) is closely related to CCHFV, sharing the same serogroup and many structural, biochemical, and cellular properties. To improve understanding of HAZV and nairovirus multiplication cycles, we developed, for the first time, a rescue system permitting efficient recovery of infectious HAZV from cDNA. This system now allows reverse genetic analysis of nairoviruses without the need for high-level biosafety containment, as is required for CCHFV. We used this system to test the importance of a DQVD caspase cleavage site exposed on the apex of the HAZV nucleocapsid protein arm domain that is cleaved during HAZV infection, for which the equivalent DEVD sequence was recently shown to be important for CCHFV growth in tick but not mammalian cells. Infectious HAZV bearing an uncleavable DQVE sequence was rescued and exhibited growth parameters equivalent to those of wild-type virus in both mammalian and tick cells, showing this site was dispensable for virus multiplication. In contrast, substitution of the DQVD motif with the similarly uncleavable AQVA sequence could not be rescued despite repeated efforts. Together, these results highlight the importance of this caspase cleavage site in the HAZV life cycle but reveal the DQVD sequence performs a critical role aside from caspase cleavage.IMPORTANCE HAZV is classified within the Nairoviridae family with CCHFV, which is one of the most lethal human pathogens in existence, requiring the highest biosafety level (BSL) containment (BSL4). In contrast, HAZV is not associated with human disease and thus can be studied using less-restrictive BSL2 protocols. Here, we report a system that is able to rescue HAZV from cDNAs, thus permitting reverse genetic interrogation of the HAZV replication cycle. We used this system to examine the role of a caspase cleavage site, DQVD, within the HAZV nucleocapsid protein that is also conserved in CCHFV. By engineering mutant viruses, we showed caspase cleavage at this site was not required for productive infection and this sequence performs a critical role in the virus life cycle aside from caspase cleavage. This system will accelerate nairovirus research due to its efficiency and utility under amenable BSL2 protocols.

Crimean-Congo hemorrhagic fever virus nucleocapsid protein harbors distinct RNA-binding sites in the stalk and head domains.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus that causes severe hemorrhagic fever with a mortality rate of up to 30% in certain outbreaks worldwide. The virus has wide endemic distribution. There is no effective antiviral therapeutic or FDA approved vaccine for this zoonotic viral illness. The multifunctional CCHFV nucleocapsid protein (N protein) plays a crucial role in the establishment of viral infection and is an important structural component of the virion. Here we show that CCHFV N protein has a distant RNA-binding site in the stalk domain that specifically recognizes the vRNA panhandle, formed by the base pairing of complementary nucleotides at the 5' and 3' termini of the vRNA genome. Using multiple approaches, including filter-bonding analysis, GFP reporter assay, and biolayer interferometry we observed an N protein-panhandle interaction both in vitro and in vivo The purified WT CCHFV N protein and the stalk domain also recognize the vRNA panhandle of hazara virus, another Nairovirus in the family Bunyaviridae, demonstrating the genus-specific nature of N protein-panhandle interaction. Another RNA-binding site was identified at the head domain of CCHFV N protein that nonspecifically recognizes the single strand RNA (ssRNA) of viral or nonviral origin. Expression of CCHFV N protein stalk domain active in panhandle binding, dramatically inhibited the hazara virus replication in cell culture, illustrating the role of N protein-panhandle interaction in Nairovirus replication. Our findings reveal the stalk domain of N protein as a potential target in therapeutic interventions to manage CCHFV disease.

Biochemical and Structural Insights into the Preference of Nairoviral DeISGylases for Interferon-Stimulated Gene Product 15 Originating from Certain Species.

UNLABELLED: The regulation of the interferon type I (IFN-I) response has been shown to rely on posttranslational modification by ubiquitin (Ub) and Ub-like interferon-stimulated gene product 15 (ISG15) to stabilize, or activate, a variety of IFN-I signaling and downstream effector proteins. Unlike Ub, which is almost perfectly conserved among eukaryotes, ISG15 is highly divergent, even among mammals. Since zoonotic viruses rely on viral proteins to recognize, or cleave, ISG15 conjugates in order to evade, or suppress, innate immunity, the impact of ISG15 biodiversity on deISGylating proteases of the ovarian tumor family (vOTU) from nairoviruses was evaluated. The enzymatic activities of vOTUs originating from the Crimean-Congo hemorrhagic fever virus, Erve virus, and Nairobi sheep disease virus were tested against ISG15s from humans, mice, shrews, sheep, bats, and camels, which are mammalian species known to be infected by nairoviruses. This along with investigation of binding by isothermal titration calorimetry illustrated significant differences in the abilities of nairovirus deISGylases to accommodate certain species of ISG15. To investigate the molecular underpinnings of species preferences of these vOTUs, a structure was determined to 2.5 Å for a complex of Erve virus vOTU protease and a mouse ISG15 domain. This structure revealed the molecular basis of Erve virus vOTU's preference for ISG15 over Ub and the first structural insight into a nonhuman ISG15. This structure also revealed key interactions, or lack thereof, surrounding three amino acids that may drive a viral deISgylase to prefer an ISG15 from one species over that of another. IMPORTANCE: Viral ovarian tumor domain proteases (vOTUs) are one of the two principal classes of viral proteases observed to reverse posttranslational modification of host proteins by ubiquitin and interferon-stimulated gene product 15 (ISG15), subsequently facilitating downregulation of IFN-I signaling pathways. Unlike the case with ubiquitin, the amino acid sequences of ISG15s from various species are notably divergent. We illustrate that vOTUs have clear preferences for ISG15s from certain species. In addition, these observations are related to the molecular insights acquired via the first X-ray structure of the vOTU from the Erve nairovirus in complex with the first structurally resolved nonhuman ISG15. This information implicates certain amino acids that drive the preference of vOTUs for ISG15s from certain species.

Modulation of Potassium Channels Inhibits Bunyavirus Infection.

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare, and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K(+)) channels to infect cells. Time of addition assays using K(+) channel modulating agents demonstrated that K(+) channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K(+) channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, two-pore domain K(+) channels (K2P) were identified as the K(+) channel family mediating BUNV K(+) channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

The molecular biology of nairoviruses, an emerging group of tick-borne arboviruses.

The nairoviruses are a rapidly emerging group of tick-borne bunyaviruses that includes pathogens of humans (Crimean-Congo hemorrhagic fever virus [CCHFV]) and livestock (Nairobi sheep disease virus [NSDV], also known as Ganjam virus), as well as a large number of viruses for which the normal vertebrate host has not been established. Studies on this group of viruses have been fairly limited, not least because CCHFV is a BSL4 human pathogen, restricting the number of labs able to study the live virus, while NSDV, although highly pathogenic in naive animals, is not seen as a threat in developed countries, making it a low priority. Nevertheless, recent years have seen significant progress in our understanding of the biology of these viruses, particularly that of CCHFV, and this article seeks to draw together our existing knowledge to generate an overall picture of their molecular biology, underlining areas of particular ignorance for future studies.

Dugbe virus ovarian tumour domain interferes with ubiquitin/ISG15-regulated innate immune cell signalling.

The ovarian tumour (OTU) domain of the nairovirus L protein has been shown to remove ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host cell proteins, which is expected to have multiple effects on cell signalling pathways. We have confirmed that the OTU domain from the L protein of the apathogenic nairovirus Dugbe virus has deubiquitinating and deISGylating activity and shown that, when expressed in cells, it is highly effective at blocking the TNF-α/NF-κB and interferon/JAK/STAT signalling pathways even at low doses. Point mutations of the catalytic site of the OTU [C40A, H151A and a double mutant] both abolished the ability of the OTU domain to deubiquitinate and deISGylate proteins and greatly reduced its effect on cell signalling pathways, confirming that it is this enzymic activity that is responsible for blocking the two signalling pathways. Expression of the inactive mutants at high levels could still block signalling, suggesting that the viral OTU can still bind to its substrate even when mutated at its catalytic site. The nairovirus L protein is a very large protein that is normally confined to the cytoplasm, where the virus replicates. When the OTU domain was prevented from entering the nucleus by expressing it as part of the N-terminal 205 kDa of the viral L protein, it continued to block type I interferon signalling, but no longer blocked the TNF-α-induced activation of NF-κB.

Inhibition of Hazara nairovirus replication by small interfering RNAs and their combination with ribavirin.

BACKGROUND: The genus Nairovirus in the family Bunyaviridae contains 34 tick-borne viruses classified into seven serogroups. Hazara virus (HAZV) belongs to the Crimean-Congo hemorrhagic fever (CCHF) serogroup that also includes CCHF virus (CCHFV) a major pathogen for humans. HAZV is an interesting model to study CCHFV due to a close serological and phylogenetical relationship and a classification which allows handling in a BSL2 laboratory. Nairoviruses are characterized by a tripartite negative-sense single stranded RNA genome (named L, M and S segments) that encode the RNA polymerase, the Gn-Gc glycoproteins and the nucleoprotein (NP), respectively. Currently, there are neither vaccines nor effective therapies for the treatment of any bunyavirus infection in humans. In this study we report, for the first time, the use of RNA interference (RNAi) as an approach to inhibit nairovirus replication. RESULTS: Chemically synthesized siRNAs were designed to target the mRNA produced by the three genomic segments. We first demonstrated that the siRNAs targeting the NP mRNA displayed a stronger antiviral effect than those complementary to the L and M transcripts in A549 cells. We further characterized the two most efficient siRNAs showing, that the induced inhibition is specific and associated with a decrease in NP synthesis during HAZV infection. Furthermore, both siRNAs depicted an antiviral activity when used before and after HAZV infection. We next showed that HAZV was sensitive to ribavirin which is also known to inhibit CCHFV. Finally, we demonstrated the additive or synergistic antiviral effect of siRNAs used in combination with ribavirin. CONCLUSIONS: Our study highlights the interest of using RNAi (alone or in combination with ribavirin) to treat nairovirus infection. This approach has to be considered for the development of future antiviral compounds targeting CCHFV, the most pathogenic nairovirus.

The high genetic variation of viruses of the genus Nairovirus reflects the diversity of their predominant tick hosts.

The genus Nairovirus (family Bunyaviridae) contains seven serogroups consisting of 34 predominantly tick-borne viruses, including several associated with severe human and livestock diseases [e.g., Crimean Congo hemorrhagic fever (CCHF) and Nairobi sheep disease (NSD), respectively]. Before this report, no comparative genetic studies or molecular detection assays had been developed for this virus genus. To characterize at least one representative from each of the seven serogroups, reverse transcriptase-polymerase chain reaction (RT-PCR) primers targeting the L polymerase-encoding region of the RNA genome of these viruses were successfully designed based on conserved amino acid motifs present in the predicted catalytic core region. Sequence analysis showed the nairoviruses to be a highly diverse group, exhibiting up to 39.4% and 46.0% nucleotide and amino acid identity differences, respectively. Virus genetic relationships correlated well with serologic groupings and with tick host associations. Hosts of these viruses include both the hard (family Ixodidae) and soft (family Argasidae) ticks. Virus phylogenetic analysis reveals two major monophyletic groups: hard tick and soft tick-vectored viruses. In addition, viruses vectored by Ornithodoros, Carios, and Argas genera ticks also form three separate monophyletic lineages. The striking similarities between tick and nairovirus phylogenies are consistent with possible coevolution of the viruses and their tick hosts. Fossil and phylogenetic data placing the hard tick-soft tick divergence between 120 and 92 million years ago suggest an ancient origin for viruses of the genus Nairovirus.

Inhibition of Dugbe nairovirus replication by human MxA protein.

Sensitivity to the interferon-induced protein, MxA, has previously been demonstrated for viruses belonging to the Orthobunyavirus, Hantavirus and Phlebovirus genera of the Bunyaviridae family. We have extended these findings to a member of the fourth and remaining genus containing viruses that infect man and other animals, the nairovirus Dugbe virus (DUGV). Indirect immunofluorescence experiments using VA9 cells (Vero cells permanently transfected with MxA cDNA) revealed strongly reduced DUGV antigen expression, suggesting that MxA inhibited DUGV replication. Western and Northern blot analyses showed significantly lower DUGV nucleocapsid (N) protein expression and DUGV genomic RNA, respectively, in the presence of MxA. Viral titres were also reduced by more than two orders of magnitude in VA9 cells compared with control VN36 cells. This finding may have application to nairovirus therapeutics.

Ultrastructural studies on the replication and morphogenesis of Nairobi sheep disease virus, a Nairovirus.

The Nairovirus Nairobi sheep disease virus (NSDV) affects sheep and goats causing severe hemorrhagic gastroenteritis and high mortality. Replication and morphogenesis of NSDV was determined by electron microscopic examination of ultra-thin sections of 143B and BHK-21 cells at varying times after infection. By 4 h post-infection (p.i.) of 143B cells, virions budding from the luminal side of the bilayer membrane of smooth membrane vesicles were observed. Morphologically mature virus particles were electron-dense, spherical and of uniform size (100 nm diameter) and accumulated in smooth membrane vesicles associated with the Golgi complex. In BHK-21 clone 13 cells, mature virus particles in smooth membrane vesicles were present by 8 h p.i. The morphogenesis of NSDV was restricted to the smooth membrane vesicles of Golgi complex, and budding of virus from other sites was not detected. Extracellular virus particles were observed by 10 h p.i., before expression of cytopathic effects. The cytopathic effects were observed at 24 h p.i. in 143B cells and at 36 h p.i. in BHK-21 cells. The morphology and morphogenesis of NSDV in BHK-21 cells and in 143B cells resembles that of other members of the family Bunyaviridae.

Bunyaviridae--natural history.

Obviously, the family Bunyaviridae is comprised of a large number of epidemiologically diverse viruses. They vary dramatically in their vector and vertebrate host relationships, geographic distributions, and epidemic potential in humans and animals. Public health practitioners, veterinarians, virologists, entomologists, biologists, ecologists, molecular biologists, and other scientists will all benefit from increased study and knowledge of this fascinating group of viruses.