A novel tamanavirus (Flaviviridae) of the European common frog (Rana temporaria) from the UK.

Flavivirids are small, enveloped, positive-sense RNA viruses from the family Flaviviridae with genomes of ~9-13 kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. However, no flavivirus-like virus has been identified in amphibians. To remedy this, we investigated the virome of the European common frog (Rana temporaria) in the UK, utilizing high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid ~11.2 kb in length. The virus encodes a single ORF of 3456 aa and 5' and 3' untranslated regions (UTRs) of 227 and 666 nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus, respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently divergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but divergent RaTV, sharing 95.64-97.39 % pairwise nucleotide identity, were also obtained from the Poole and Deal samples, indicating that RaTV is widespread in UK frog samples. Bioinformatic analyses of predicted secondary structures in the 3'UTR of RaTV showed the presence of an exoribonuclease-resistant RNA (xrRNA) structure standard in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro Xrn1 digestion assay showing that RaTV probably forms a functional Xrn1-resistant xrRNA.

Inactivation of SARS-CoV-2 infectivity in platelet concentrates or plasma following treatment with ultraviolet C light or with methylene blue combined with visible light.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unlikely to be a major transfusion-transmitted pathogen; however, convalescent plasma is a treatment option used in some regions. The risk of transfusion-transmitted infections can be minimized by implementing Pathogen Inactivation (PI), such as THERAFLEX MB-plasma and THERAFLEX UV-Platelets systems. Here we examined the capability of these PI systems to inactivate SARS-CoV-2. STUDY DESIGN AND METHODS: SARS-CoV-2 spiked plasma units were treated using the THERAFLEX MB-Plasma system in the presence of methylene blue (~0.8 µmol/L; visible light doses: 20, 40, 60, and 120 [standard] J/cm2 ). SARS-CoV-2 spiked platelet concentrates (PCs) were treated using the THERAFLEX UV-platelets system (UVC doses: 0.05, 0.10, 0.15, and 0.20 [standard] J/cm2 ). Samples were taken prior to the first and after each illumination dose, and viral infectivity was assessed using an immunoplaque assay. RESULTS: Treatment of spiked plasma with the THERAFLEX MB-Plasma system resulted in an average ≥5.03 log10 reduction in SARS-CoV-2 infectivity at one third (40 J/cm2 ) of the standard visible light dose. For the platelet concentrates (PCs), treatment with the THERAFLEX UV-Platelets system resulted in an average ≥5.18 log10 reduction in SARS-CoV-2 infectivity at the standard UVC dose (0.2 J/cm2 ). CONCLUSIONS: SARS-CoV-2 infectivity was reduced in plasma and platelets following treatment with the THERAFLEX MB-Plasma and THERAFLEX UV-Platelets systems, to the limit of detection, respectively. These PI technologies could therefore be an effective option to reduce the risk of transfusion-transmitted emerging pathogens.

Ancestral SARS-CoV-2, but not Omicron, replicates less efficiently in primary pediatric nasal epithelial cells.

Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air-liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.

Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 by Disrupting the Spike-ACE2 Interaction.

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19.

No evidence of human genome integration of SARS-CoV-2 found by long-read DNA sequencing.

A recent study proposed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we apply deep (>50×) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2 and do not find the virus integrated into the genome. By examining ONT data from separate HEK293T cultivars, we completely resolve 78 L1 insertions arising in vitro in the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV)-positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions by ONT sequencing. That we find no evidence of SARS-CoV-2 integration suggests that such events are, at most, extremely rare in vivo and therefore are unlikely to drive oncogenesis or explain post-recovery detection of the virus.

Neuroinvasiveness of the MR766 strain of Zika virus in IFNAR-/- mice maps to prM residues conserved amongst African genotype viruses.

Zika virus (ZIKV) strains are classified into the African and Asian genotypes. The higher virulence of the African MR766 strain, which has been used extensively in ZIKV research, in adult IFNα/ß receptor knockout (IFNAR-/-) mice is widely viewed as an artifact associated with mouse adaptation due to at least 146 passages in wild-type suckling mouse brains. To gain insights into the molecular determinants of MR766's virulence, a series of genes from MR766 were swapped with those from the Asian genotype PRVABC59 isolate, which is less virulent in IFNAR-/- mice. MR766 causes 100% lethal infection in IFNAR-/- mice, but when the prM gene of MR766 was replaced with that of PRVABC59, the chimera MR/PR(prM) showed 0% lethal infection. The reduced virulence was associated with reduced neuroinvasiveness, with MR766 brain titers ≈3 logs higher than those of MR/PR(prM) after subcutaneous infection, but was not significantly different in brain titers of MR766 and MR/PR(prM) after intracranial inoculation. MR/PR(prM) also showed reduced transcytosis when compared with MR766 in vitro. The high neuroinvasiveness of MR766 in IFNAR-/- mice could be linked to the 10 amino acids that differ between the prM proteins of MR766 and PRVABC59, with 5 of these changes affecting positive charge and hydrophobicity on the exposed surface of the prM protein. These 10 amino acids are highly conserved amongst African ZIKV isolates, irrespective of suckling mouse passage, arguing that the high virulence of MR766 in adult IFNAR-/- mice is not the result of mouse adaptation.

Chimeric viruses of the insect-specific flavivirus Palm Creek with structural proteins of vertebrate-infecting flaviviruses identify barriers to replication of insect-specific flaviviruses in vertebrate cells.

Here we report the generation of novel chimeric flaviviruses, which express the prM and E proteins of either dengue or Zika viruses on the genomic backbone of Palm Creek virus (PCV), an insect-specific flavivirus. The chimeric virus particles were antigenically indistinguishable from their parental prM-E donors, but were unable to infect vertebrate cells. An additional chimera (PCV structural genes in the backbone of West Nile virus - WNV/PCV-prME) was also unable to infect vertebrate cells, but transfection with RNA from this virus resulted in detectable RNA replication and translation but no infectious virion production. These data suggest multiple blocks at the entry, RNA replication and assembly/release stages of insect-specific flavivirus (ISF) infection in vertebrate cells. Serial passaging of these chimeric viruses in mosquito cells identified amino acid substitutions that may lead to increased replication efficiency. These chimeric viruses provide unique tools to further dissect the mechanisms of the host restriction of ISFs.

Determinants of Zika virus host tropism uncovered by deep mutational scanning.

Arboviruses cycle between, and replicate in, both invertebrate and vertebrate hosts, which for Zika virus (ZIKV) involves Aedes mosquitoes and primates1. The viral determinants required for replication in such obligate hosts are under strong purifying selection during natural virus evolution, making it challenging to resolve which determinants are optimal for viral fitness in each host. Herein we describe a deep mutational scanning (DMS) strategy2-5 whereby a viral cDNA library was constructed containing all codon substitutions in the C-terminal 204 amino acids of ZIKV envelope protein (E). The cDNA library was transfected into C6/36 (Aedes) and Vero (primate) cells, with subsequent deep sequencing and computational analyses of recovered viruses showing that substitutions K316Q and S461G, or Q350L and T397S, conferred substantial replicative advantages in mosquito and primate cells, respectively. A 316Q/461G virus was constructed and shown to be replication-defective in mammalian cells due to severely compromised virus particle formation and secretion. The 316Q/461G virus was also highly attenuated in human brain organoids, and illustrated utility as a vaccine in mice. This approach can thus imitate evolutionary selection in a matter of days and identify amino acids key to the regulation of virus replication in specific host environments.

CCR2 Plays a Protective Role in Rocio Virus-Induced Encephalitis by Promoting Macrophage Infiltration Into the Brain.

Rocio virus (ROCV) is a highly neuropathogenic mosquito-transmitted flavivirus responsible for an unprecedented outbreak of human encephalitis during 1975-1976 in Sao Paulo State, Brazil. Previous studies have shown an increased number of inflammatory macrophages in the central nervous system (CNS) of ROCV-infected mice, implying a role for macrophages in the pathogenesis of ROCV. Here, we show that ROCV infection results in increased expression of CCL2 in the blood and in infiltration of macrophages into the brain. Moreover, we show, using CCR2 knockout mice, that CCR2 expression is essential for macrophage infiltration in the brain during ROCV infection and that the lack of CCR2 results in increased disease severity and mortality. Thus, our findings show the protective role of CCR2-mediated infiltration of macrophages in the brain during ROCV infection.

A vaccinia-based single vector construct multi-pathogen vaccine protects against both Zika and chikungunya viruses.

Zika and chikungunya viruses have caused major epidemics and are transmitted by Aedes aegypti and/or Aedes albopictus mosquitoes. The "Sementis Copenhagen Vector" (SCV) system is a recently developed vaccinia-based, multiplication-defective, vaccine vector technology that allows manufacture in modified CHO cells. Herein we describe a single-vector construct SCV vaccine that encodes the structural polyprotein cassettes of both Zika and chikungunya viruses from different loci. A single vaccination of mice induces neutralizing antibodies to both viruses in wild-type and IFNAR-/- mice and protects against (i) chikungunya virus viremia and arthritis in wild-type mice, (ii) Zika virus viremia and fetal/placental infection in female IFNAR-/- mice, and (iii) Zika virus viremia and testes infection and pathology in male IFNAR-/- mice. To our knowledge this represents the first single-vector construct, multi-pathogen vaccine encoding large polyproteins, and offers both simplified manufacturing and formulation, and reduced "shot burden" for these often co-circulating arboviruses.

Genetic characterization of Cacipacoré virus from ticks collected in São Paulo State, Brazil.

Cacipacoré virus (CPCV) is a potential emerging virus classified in the genus Flavivirus, family Flaviviridae. In the present study, we present the genetic characterization of a CPCV isolated from ticks (Amblyomma cajennense) collected from a sick capybara (Hydrochoerus hydrochaeris) in São Paulo State, Brazil. The CPCV isolate shares the typical genomic organization of flaviviruses with 10,857 nucleotides in length and a single open reading frame of 10,284 nucleotides encoding a polyprotein of 3,427 amino acids. Phylogenetic analysis revealed that CPCV is unique, as a potentially tick-borne virus, in the Japanese encephalitis virus serogroup.

The I22V and L72S substitutions in West Nile virus prM protein promote enhanced prM/E heterodimerisation and nucleocapsid incorporation.

BACKGROUND: Amino acid substitutions I22V and L72S in the prM protein of West Nile virus Kunjin strain (WNVKUN) were previously shown to enhance virus secretion and virulence, but a mechanism by which this occurred was not determined. FINDINGS: Using pulse-chase experiments followed by co-immunoprecipitation with anti-E antibody, we demonstrated that the I22V and L72S substitutions enhanced prM/E heterodimerization for both the E-glycosylated and E-unglycosylated virus. Furthermore, analysis of secreted particles revealed that I22V and L72S substitutions also enhanced nucleocapsid incorporation. CONCLUSIONS: We have demonstrated mechanistically that improved secretion of virus particles in the presence of I22V and L72S substitutions was contributed by more efficient prM/E heterodimerization.

Post-translational regulation and modifications of flavivirus structural proteins.

Flaviviruses are a group of single-stranded, positive-sense RNA viruses that generally circulate between arthropod vectors and susceptible vertebrate hosts, producing significant human and veterinary disease burdens. Intensive research efforts have broadened our scientific understanding of the replication cycles of these viruses and have revealed several elegant and tightly co-ordinated post-translational modifications that regulate the activity of viral proteins. The three structural proteins in particular - capsid (C), pre-membrane (prM) and envelope (E) - are subjected to strict regulatory modifications as they progress from translation through virus particle assembly and egress. The timing of proteolytic cleavage events at the C-prM junction directly influences the degree of genomic RNA packaging into nascent virions. Proteolytic maturation of prM by host furin during Golgi transit facilitates rearrangement of the E proteins at the virion surface, exposing the fusion loop and thus increasing particle infectivity. Specific interactions between the prM and E proteins are also important for particle assembly, as prM acts as a chaperone, facilitating correct conformational folding of E. It is only once prM/E heterodimers form that these proteins can be secreted efficiently. The addition of branched glycans to the prM and E proteins during virion transit also plays a key role in modulating the rate of secretion, pH sensitivity and infectivity of flavivirus particles. The insights gained from research into post-translational regulation of structural proteins are beginning to be applied in the rational design of improved flavivirus vaccine candidates and make attractive targets for the development of novel therapeutics.

West Nile virus genome with glycosylated envelope protein and deletion of alpha helices 1, 2, and 4 in the capsid protein is noninfectious and efficiently secretes subviral particles.

Flavivirus genomes with deletions in the capsid (C) gene are attractive vaccine candidates, as they secrete highly immunogenic subviral particles (SVPs) without generating infectious virus. Here, we report that cytomegalovirus promoter-driven cDNA of West Nile virus Kunjin (KUNV) containing a glycosylation motif in the envelope (E) gene and a combined deletion of alpha helices 1, 2, and 4 in C produces significantly more SVPs than KUNV cDNAs with nonglycosylated E and various other deletions in C.

Interferon response factors 3 and 7 protect against Chikungunya virus hemorrhagic fever and shock.

Chikungunya virus (CHIKV) infections can produce severe disease and mortality. Here we show that CHIKV infection of adult mice deficient in interferon response factors 3 and 7 (IRF3/7(-/-)) is lethal. Mortality was associated with undetectable levels of alpha/beta interferon (IFN-α/ß) in serum, ∼50- and ∼10-fold increases in levels of IFN-γ and tumor necrosis factor (TNF), respectively, increased virus replication, edema, vasculitis, hemorrhage, fever followed by hypothermia, oliguria, thrombocytopenia, and raised hematocrits. These features are consistent with hemorrhagic shock and were also evident in infected IFN-α/ß receptor-deficient mice. In situ hybridization suggested CHIKV infection of endothelium, fibroblasts, skeletal muscle, mononuclear cells, chondrocytes, and keratinocytes in IRF3/7(-/-) mice; all but the latter two stained positive in wild-type mice. Vaccination protected IRF3/7(-/-) mice, suggesting that defective antibody responses were not responsible for mortality. IPS-1- and TRIF-dependent pathways were primarily responsible for IFN-α/ß induction, with IRF7 being upregulated >100-fold in infected wild-type mice. These studies suggest that inadequate IFN-α/ß responses following virus infection can be sufficient to induce hemorrhagic fever and shock, a finding with implications for understanding severe CHIKV disease and dengue hemorrhagic fever/dengue shock syndrome.

Determinants of attenuation in the envelope protein of the flavivirus Alfuy.

Murray Valley encephalitis virus (MVEV) is a mosquito-borne flavivirus endemic to Australia and Papua New Guinea. Most strains of MVEV cause potentially fatal cases of encephalitis in humans and horses, and have been shown to be highly neuroinvasive in weanling mice. In contrast, the naturally occurring subtype Alfuy virus (ALFV) has never been associated with human disease, nor is it neuroinvasive in weanling mice, even at high doses. To identify viral factors associated with ALFV attenuation, a chimeric infectious clone was constructed containing the structural genes premembrane (prM) and envelope (E) of ALFV swapped into the MVEV genome. The resulting virus (vMVEV/ALFVstr) was no longer neuroinvasive in mice, suggesting that motifs within prM-E of ALFV confer attenuation. To define these motifs further, mutants were constructed by targeting divergent sequences between the MVEV and ALFV E proteins that are known markers of virulence in other encephalitic flaviviruses. MVEV mutants containing a unique ALFV sequence in the flexible hinge region (residues 273-277) or lacking the conserved glycosylation site at position 154 were significantly less neuroinvasive in mice than wild-type MVEV, as determined by delayed time to death or increased LD(50). Conversely, when the corresponding MVEV sequences were inserted into the vMVEV/ALFVstr chimera, the mutant containing the MVEV hinge sequence was more neuroinvasive than the parental chimera, though not to the same level as wild-type MVEV. These results identify the hinge region and E protein glycosylation as motifs that contribute to the attenuation of ALFV.

pDNA-lipoplexes engrafted with flagellin-related peptide induce potent immunity and anti-tumour effects.

Complexes of cationic lipids and DNA (lipoplexes) are widely used for non-viral gene delivery and DNA vaccine development, but cationic lipids are toxic and promote non-specific interactions with cells, leading to poor efficacy. Near-neutral lipoplexes, on the other hand, can obviate toxicity, but a convenient means to target them to specific cells such as dendritic cells (DCs) has been lacking. Here, we show that a His-tagged flagellin-derived peptide (denoted 9Flg), previously reported to promote binding of liposomal antigen to TLR5-expressing cells, can be used to target near-neutral pDNA-lipoplexes incorporating the chelator lipid NTA(3)-DTDA (3(nitrilotriacetic acid)-ditetradecylamine) to DCs and other antigen-presenting cells (APCs). Thus, we show that pDNA-lipoplexes engrafted with 9Flg target pDNA to APCs in vitro and in vivo. Following i.v. administration, radiolabelled 9Flg-lipoplexes exhibited increased accumulation in spleen, lung and liver. Vaccination of C57BL/6 mice with 9Flg-lipoplexes containing either pcDNA3.1-SIIN (pSIIN) or a Kunjin virus replicon-based vector (pKUN), each encoding the epitope OVA(257-264) (SIINFEKL), induced Ag-specific T cell priming, and elicited strong cellular immunity as reflected by a marked increase in the number of Ag-responsive IFN-γ-producing CD8(+) T cells. Importantly, compared to i.m. injection of these SIINFEKL-encoding pDNAs in naked form, the i.v. administration of pSIIN or pKUN in 9Flg-lipoplexes to C57BL/6 mice induced a significantly more potent anti-tumour response in the B16-OVA melanoma tumour model. The targeting of near-neutral 9Flg-lipoplexes bearing pDNA encoding tumour antigens to TLR5 on APCs, therefore, is a powerful approach for developing more effective DNA vaccines and immunotherapies.

Kunjin replicon-based simian immunodeficiency virus gag vaccines.

An RNA-based, non-cytopathic replicon vector system, based on the flavivirus Kunjin, has shown considerable promise as a new vaccine delivery system. Here we describe the testing in mice of four different SIVmac239 gag vaccines delivered by Kunjin replicon virus-like-particles. The four vaccines encoded the wild type gag gene, an RNA-optimised gag gene, a codon-optimised gag gene and a modified gag-pol gene construct. The vaccines behaved quite differently for induction of effector memory and central memory responses, for mediation of protection, and with respect to insert stability, with the SIV gag-pol vaccine providing the optimal performance. These results illustrate that for an RNA-based vector the RNA sequence of the antigen can have profound and unforeseen consequences on vaccine behaviour.

Cholesterol manipulation by West Nile virus perturbs the cellular immune response.

Complex membrane structures induced by West Nile virus (WNV), an enveloped RNA virus, are required for efficient viral replication. How these membranes are induced and how they facilitate the viral life cycle are unknown. We show that WNV modulates host cell cholesterol homeostasis by upregulating cholesterol biosynthesis and redistributing cholesterol to viral replication membranes. Manipulating cholesterol levels and altering concentrations of cellular geranylgeranylated proteins had a deleterious effect on virus replication. Depletion of the key cholesterol-synthesizing enzyme 3-hydroxy-methyglutaryl-CoA reductase drastically hampered virus replication. Significantly, virus-induced redistribution of cellular cholesterol downregulated the interferon-stimulated Jak-STAT antiviral signaling response to infection. This defect could be partially restored by exogenous addition of cholesterol, which increased the ability of infected cells to respond to interferon. We propose that, by manipulating cellular cholesterol, WNV utilizes the cellular response to cholesterol deficiency and dependence of antiviral signaling pathways on cholesterol-rich microdomains to facilitate viral replication and survival.

Crystal structure and activity of Kunjin virus NS3 helicase; protease and helicase domain assembly in the full length NS3 protein.

Flaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-triphosphatase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication. In addition, NS3 assists the initiation of replication by unwinding the RNA secondary structure in the 3' non-translated region (NTR). We report here the three-dimensional structure (at 3.1 A resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin virus, an Australian variant of the West Nile virus. As for homologous helicases, NS3:186-619 is composed of three domains, two of which are structurally related and held to host the NTPase and RTPase active sites. The third domain (C-terminal) is involved in RNA binding/recognition. The NS3:186-619 construct occurs as a dimer in solution and in the crystals. We show that NS3:186-619 displays both ATPase and RTPase activities, that it can unwind a double-stranded RNA substrate, being however inactive on a double-stranded DNA substrate. Analysis of different constructs shows that full length NS3 displays increased helicase activity, suggesting that the protease domain plays an assisting role in the RNA unwinding process. The structural interaction between the helicase and protease domain has been assessed using small angle X-ray scattering on full length NS3, disclosing that the protease and helicase domains build a rather elongated molecular assembly differing from that observed in the NS3 protein from hepatitis C virus.