Comparative Pathogenesis of Two Lineages of Powassan Virus Reveals Distinct Clinical Outcome, Neuropathology, and Inflammation.

Tick-borne flaviviruses (TBFV) can cause severe neuroinvasive disease which may result in death or long-term neurological deficit in over 50% of survivors. Multiple mechanisms for invasion of the central nervous system (CNS) by flaviviruses have been proposed including axonal transport, transcytosis, endothelial infection, and Trojan horse routes. Flaviviruses may utilize different or multiple mechanisms of neuroinvasion depending on the specific virus, infection site, and host variability. In this work we have shown that the infection of BALB/cJ mice with either Powassan virus lineage I (Powassan virus) or lineage II (deer tick virus) results in distinct spatial tropism of infection in the CNS which correlates with unique clinical presentations for each lineage. Comparative transcriptomics of infected brains demonstrates the activation of different immune pathways and downstream host responses. Ultimately, the comparative pathology and transcriptomics are congruent with different clinical signs in a murine model. These results suggest that the different disease presentations occur in clinical cases due to the inherent differences in the two lineages of Powassan virus.

Strain-Dependent Assessment of Powassan Virus Transmission to Ixodes scapularis Ticks.

Powassan virus (POWV) is an emerging tick-borne encephalitic virus in Lyme disease-endemic sites in North America. Due to range expansion and local intensification of blacklegged tick vector (Ixodes scapularis) populations in the northeastern and upper midwestern U.S., human encephalitis cases are increasingly being reported. A better understanding of the transmission cycle between POWV and ticks is required in order to better predict and understand their public health burden. Recent phylogeographic analyses of POWV have identified geographical structuring, with well-defined northeastern and midwestern clades of the lineage II subtype. The extent that geographic and genetically defined sublineages differ in their ability to infect and be transmitted by blacklegged ticks is unclear. Accordingly, we determined whether there are strain-dependent differences in the transmission of POWV to ticks at multiple life stages. Five recent, low-passage POWV isolates were used to measure aspects of vector competence, using viremic and artificial infection methods. Infection rates in experimental ticks remained consistent between all five isolates tested, resulting in a 12-20% infection rate and some differences in viral load. We confirm that these differences are likely not due to differences in host viremia. Our results demonstrate that blacklegged ticks are susceptible to, and capable of transmitting, all tested strains and suggest that the tick-virus association is stable across diverse viral genotypes.

No detection of tick-borne encephalitis virus RNA in blood, urine or saliva of hospitalised immunocompetent tick-borne encephalitis patients.

Tick-borne encephalitis (TBE) is usually diagnosed based on the presence of TBE virus (TBEV)-specific IgM and IgG antibodies in serum. However, antibodies induced by vaccination or cross-reactivity to previous flavivirus infections may result in false positive TBEV serology. Detection of TBEV RNA may be an alternative diagnostic approach to detect viral presence and circumvent the diagnostic difficulties present when using serology. Viral RNA in blood is commonly detectable only in the first viremic phase usually lasting up to two weeks, and not in the second neurologic phase, when the patients contact the health care system and undergo diagnostic work-up. TBEV RNA has previously been detected in urine in a few retrospective TBE cases in the neurologic phase, and furthermore RNA of other flaviviruses has been detected in patient saliva. In this study, blood, saliva and urine were collected from 31 hospitalised immunocompetent patients with pleocytosis and symptoms of aseptic meningitis and/or encephalitis, suspected to have TBE. We wanted to pursue if molecular testing of TBEV RNA in these patient materials may be useful in the diagnostics. Eleven of the 31 study patients were diagnosed with TBE based on ELISA detection of TBEV specific IgG and IgM antibodies. None of the study patients had TBEV RNA detectable in any of the collected patient material.

Interspecies co-feeding transmission of Powassan virus between a native tick, Ixodes scapularis, and the invasive East Asian tick, Haemaphysalis longicornis.

BACKGROUND: Powassan virus, a North American tick-borne flavivirus, can cause severe neuroinvasive disease in humans. While Ixodes scapularis are the primary vectors of Powassan virus lineage II (POWV II), also known as deer tick virus, recent laboratory vector competence studies showed that other genera of ticks can horizontally and vertically transmit POWV II. One such tick is the Haemaphysalis longicornis, an invasive species from East Asia that recently established populations in the eastern USA and already shares overlapping geographic range with native vector species such as I. scapularis. Reports of invasive H. longicornis feeding concurrently with native I. scapularis on multiple sampled hosts highlight the potential for interspecies co-feeding transmission of POWV II. Given the absence of a clearly defined vertebrate reservoir host for POWV II, it is possible that this virus is sustained in transmission foci via nonviremic transmission between ticks co-feeding on the same vertebrate host. The objective of this study was to evaluate whether uninfected H. longicornis co-feeding in close proximity to POWV II-infected I. scapularis can acquire POWV independent of host viremia. METHODS: Using an in vivo tick transmission model, I. scapularis females infected with POWV II ("donors") were co-fed on mice with uninfected H. longicornis larvae and nymphs ("recipients"). The donor and recipient ticks were infested on mice in various sequences, and mouse infection status was monitored by temporal screening of blood for POWV II RNA via quantitative reverse transcription polymerase chain reaction (q-RT-PCR). RESULTS: The prevalence of POWV II RNA was highest in recipient H. longicornis that fed on viremic mice. However, nonviremic mice were also able to support co-feeding transmission of POWV, as demonstrated by the detection of viral RNA in multiple H. longicornis dispersed across different mice. Detection of viral RNA at the skin site of tick feeding but not at distal skin sites indicates that a localized skin infection facilitates transmission of POWV between donor and recipient ticks co-feeding in close proximity. CONCLUSIONS: This is the first report examining transmission of POWV between co-feeding ticks. Against the backdrop of multiple unknowns related to POWV ecology, findings from this study provide insight on possible mechanisms by which POWV could be maintained in nature.

In vivo rescue of arboviruses directly from subgenomic DNA fragments.

Reverse genetic systems are mainly used to rescue recombinant viral strains in cell culture. These tools have also been used to generate, by inoculating infectious clones, viral strains directly in living animals. We previously developed the "Infectious Subgenomic Amplicons" (ISA) method, which enables the rescue of single-stranded positive sense RNA viruses in vitro by transfecting overlapping subgenomic DNA fragments. Here, we provide proof-of-concept for direct in vivo generation of infectious particles following the inoculation of subgenomic amplicons. First, we rescued a strain of tick-borne encephalitis virus in mice to transpose the ISA method in vivo. Subgenomic DNA fragments were amplified using a 3-fragment reverse genetics system and inoculated intramuscularly. Almost all animals were infected when quantities of DNA inoculated were at least 20 µg. We then optimized our procedure in order to increase the animal infection rate. This was achieved by adding an electroporation step and/or using a simplified 2- fragment reverse genetics system. Under optimal conditions, a large majority of animals were infected with doses of 20 ng of DNA. Finally, we demonstrated the versatility of this method by applying it to Japanese encephalitis and Chikungunya viruses. This method provides an efficient strategy for in vivo rescue of arboviruses. Furthermore, in the context of the development of DNA-launched live attenuated vaccines, this new approach may facilitate the generation of attenuated strains in vivo. It also enables to deliver a substance free of any vector DNA, which seems to be an important criterion for the development of human vaccines.

Fatal tick-borne encephalitis virus infection in Dalmatian puppy-dogs after putative vector independent transmission.

In a retrospective metatranscriptomics study, we identified tick-borne encephalitis virus (TBEV) to be the causative agent for a fatal non-suppurative meningoencephalitis in a three-week-old Dalmatian puppy in Switzerland. Further investigations showed that the two other littermates with similar signs and pathological lesions were also positive for TBEV. By using an unbiased approach of combining high-throughput sequencing (HTS) and bioinformatics we were able to solve the etiology and discover an unusual case of TBEV in three young puppies. Based on our findings, we suggest that a vector-independent transmission of TBEV occurred and that most likely an intrauterine infection led to the severe and fulminant disease of the entire litter. We were able to demonstrate the presence of TBEV RNA by in situ hybridization (ISH) in the brain of all three puppies. Furthermore, we were able to detect TBEV by RT-qPCR in total RNA extracted from formalin-fixed and paraffin embedded (FFPE) blocks containing multiple peripheral organs. Overall, our findings shed light on alternative vector-independent transmission routes of TBEV infections in dogs and encourage veterinary practitioners to consider TBEV as an important differential diagnosis in neurological cases in dogs.

Identification of New Microfoci and Genetic Characterization of Tick-Borne Encephalitis Virus Isolates from Eastern Germany and Western Poland.

(1) Background: Tick-borne encephalitis (TBE) is the most important tick-borne viral disease in Eurasia, although effective vaccines are available. Caused by the tick-borne encephalitis virus (TBEV, syn. Orthoflavivirus encephalitidis), in Europe, it is transmitted by ticks like Ixodes ricinus and Dermacentor reticulatus. TBEV circulates in natural foci, making it endemic to specific regions, such as southern Germany and northeastern Poland. Our study aimed to identify new TBEV natural foci and genetically characterize strains in ticks in previously nonendemic areas in Eastern Germany and Western Poland. (2) Methods: Ticks were collected from vegetation in areas reported by TBE patients. After identification, ticks were tested for TBEV in pools of a maximum of 10 specimens using real-time RT-PCR. From the positive TBEV samples, E genes were sequenced. (3) Results: Among 8400 ticks from 19 sites, I. ricinus (n = 4784; 56.9%) was predominant, followed by D. reticulatus (n = 3506; 41.7%), Haemaphysalis concinna (n = 108; 1.3%), and I. frontalis (n = 2; <0.1%). TBEV was detected in 19 pools originating in six sites. The phylogenetic analyses revealed that TBEV strains from Germany and Poland clustered with other German strains, as well as those from Finland and Estonia. (4) Conclusions: Although there are still only a few cases are reported from these areas, people spending much time outdoors should consider TBE vaccination.

The First Record of Omsk Hemorrhagic Fever Virus and Tick-Borne Encephalitis Virus of Baltic Lineage from the Kemerovo Region of Russia.

Objectives: Tick-borne encephalitis virus Siberian subtype (TBEV-Sib) and Omsk hemorrhagic fever virus (OHFV) are causative agents of natural focal infections in Western Siberia, Russia. The distribution of TBEV phylogenetic lineages and OHFV in the Kemerovo Region of Western Siberia remains poorly investigated. Methods: The phylogenetic analyses of fragment genome sequences 26 flaviviruses identified in 2019 were performed, and the amino acid variation was determined to reveal to which clusteron they belong. The age of Baltic and Asian lineages of the TBEV-Sib was calculated for Kemerovo District and Region, respectively. Results: Twenty-five isolates were members of three TBEV-Sib phylogenetic lineages: Baltic (48%), Asian (36%), and East Siberian (16%). The Baltic lineage's eastern boundary is commonly thought to be in the Novosibirsk Region, but our data suggest that it may reach further east. Analysis of the Baltic lineage clusteron structure showed that the isolates found are unique (6) or belong to clusteron-founder 3D (1) and derived clusteron 3O (5). Based on the age of 3O clusteron, Baltic lineage could have appeared in the Kemerovo Region by the late 1970s. One of the isolated viruses turned out to be the OHFV of the first subtype and not to belong to any known clusteron. This finding is the first known detection of the virus outside the endemic area of Russia. Given the recent discovery of OHFV in Kazakhstan, it can be assumed that the area of this virus distribution is much wider than previously thought. Conclusions: This report provides insights into the population structure of TBEV and OHFV, which may be helpful for epidemiological investigation and surveillance of the viruses.

Studies on the sequential pathology of Kyasanur Forest Disease (KFD) in Mouse brain: KFD virus induces apoptosis of neurons in cerebrum and hippocampus.

The sequential pathology of Kyasanur forest disease (KFD) in mouse brain was assessed in this study. Kyasanur forest disease virus (KFDV) strain P9605 used in this study was confirmed by real-time reverse transcriptase-polymerase chain reaction targeting the NS5 gene. Mouse Lethal Dose 50 (MLD50) of the virus was determined by in-vivo mice inoculation test. One MLD50 of the KFDV was inoculated intra-cerebrally into 36 mice aged 2-3 weeks. Another group of 36 age-matched mice that served as control group were inoculated with plain media. Six mice each from infected and control groups were euthanized every 24 hrs intervals for six days. Brain tissues were collected in 10% NBF. The collected brain tissues were processed and subjected to histopathological studies by Hematoxylin and Eosin staining. Grossly, the infected mice showed symptoms of dullness, hunched back appearance, weakness, sluggish movements with indication of hind quarter paralysis on day four post-infection. These symptoms got aggravated with complete paralysis of the hind quarters, inability to move and death on 5th and 6th day post-infection. Microscopically, the brain showed apoptosis of neurons, perivascular cuffing, gliosis, congestion, neuropil vacuolation, meningitis, degeneration, and necrotic neurons. The real-time RT-PCR on hippocampus of the KFDV-infected mouse brain showed three-fold higher expression levels of Caspase 3, a crucial mediator of apoptosis. The cerebral cortex, cerebellum and hippocampus that control the motor neuron activities and muscle tone were primarily affected, possibly correlating with the gross symptoms of hind quarter paralysis, ataxia, and other motor neuron dysfunctions noticed. Taken together, these findings reveal that KFDV induces apoptosis of neurons in the cerebrum and hippocampus of KFDV infected mice. Further studies are needed to confirm if the lesions noticed in mice brain simulate the brain lesions in humans since gross motor-neuron symptoms are similar in mice as well as humans.

The Prevalence of Tick-Borne Encephalitis Virus in Wild Rodents Captured in Tick-Borne Encephalitis Foci in Highly Endemic Lithuania.

Wild rodents are considered to be one of the most important TBEV-amplifying reservoir hosts; therefore, they may be suitable for foci detection studies. To investigate the effectiveness of viral RNA detection in wild rodents for suspected TBEV foci confirmation, we trapped small rodents (n = 139) in various locations in Lithuania where TBEV was previously detected in questing ticks. Murine neuroblastoma Neuro-2a cells were inoculated with each rodent sample to maximize the chances of detecting viral RNA in rodent samples. TBEV RNA was detected in 74.8% (CI 95% 66.7-81.1) of the brain and/or internal organ mix suspensions, and the prevalence rate increased significantly following sample cultivation in Neuro-2a cells. Moreover, a strong correlation (r = 0.88; p < 0.05) was found between the average monthly air temperature of rodent trapping and the TBEV RNA prevalence rate in cell culture isolates of rodent suspensions, which were PCR-negative before cultivation in cell culture. This study shows that wild rodents are suitable sentinel animals to confirm TBEV foci. In addition, the study results demonstrate that sample cultivation in cell culture is a highly efficient method for increasing TBEV viral load to detectable quantities.

Direct Evidence of Powassan Virus Vertical Transmission in Ixodes scapularis in Nature.

Powassan virus (POWV) is a tick-borne flavivirus endemic in North America and Russia. Experimental infections with POWV have confirmed horizontal, transstadial, vertical, and cofeeding transmission routes for potential virus maintenance. In the field, vertical transmission has never been observed. During New York State tick-borne pathogen surveillance, POWV RNA and/or infectious POWV was detected in five pools of questing Ixodes scapularis larvae. Additionally, engorged female I. scapularis adults were collected from hunter-harvested white-tailed deer (Odocoileus virginianus) in a region with relatively high tick infection rates of POWV and allowed to oviposit under laboratory conditions. POWV RNA was detected in three female adult husks and one pool of larvae from a positive female. Infectious virus was isolated from all three RNA-positive females and the single positive larval pool. The detection of RNA and infectious virus in unfed questing larvae from the field and larvae from replete females collected from the primary tick host implicates vertical transmission as a potential mechanism for the maintenance of POWV in I. scapularis in nature, and elucidates the potential epidemiological significance of larval ticks in the transmission of POWV to humans.

Differential susceptibility of geographically distinct Ixodes ricinus populations to tick-borne encephalitis virus and louping ill virus.

Tick-borne encephalitis virus (TBEV) is an emerging pathogen in the Netherlands. Multiple divergent viral strains are circulating and the focal distribution of TBEV remains poorly understood. This may, however, be explained by differences in the susceptibility of tick populations for specific viruses and viral strains, and by viral strains having higher infection success in their local tick population. We investigated this hypothesis by exposing Dutch Ixodes ricinus ticks to two different TBEV strains: TBEV-NL from the Netherlands and TBEV-Neudoerfl from Austria. In addition, we exposed ticks to louping Ill virus (LIV), which is endemic to large parts of the United Kingdom and Ireland, but has not been reported in the Netherlands. Ticks were collected from two locations in the Netherlands: one location without evidence of TBEV circulation and one location endemic for the TBEV-NL strain. Ticks were infected in a biosafety level 3 laboratory using an artificial membrane feeding system. Ticks collected from the region without evidence of TBEV circulation had lower infection rates for TBEV-NL as compared to TBEV-Neudoerfl. Vice versa, ticks collected from the TBEV-NL endemic region had higher infection rates for TBEV-NL compared to TBEV-Neudoerfl. In addition, LIV infection rates were much lower in Dutch ticks compared to TBEV, which may explain why LIV is not present in the Netherlands. Our findings show that ticks from two distinct geographical populations differ in their susceptibility to TBEV strains, which could be the result of differences in the genetic background of the tick populations.

Recapitulation of the Powassan virus life cycle in cell culture.

Powassan virus (POWV) is a tick-borne flavivirus known for causing fatal neuroinvasive diseases in humans. Recently, there has been a noticeable increase in POWV infections, emphasizing the urgency of understanding viral replication, pathogenesis, and developing interventions. Notably, there are no approved vaccines or therapeutics for POWV, and its classification as a biosafety level-3 (BSL-3) agent hampers research. To overcome these obstacles, we developed a replicon system, a self-replicating RNA lacking structural proteins, making it safe to operate in a BSL-2 environment. We constructed a POWV replicon carrying the Gaussia luciferase (Gluc) reporter gene and blasticidin (BSD) selectable marker. Continuous BSD selection led to obtain a stable POWV replicon-carrying Huh7 cell lines. We identified cell culture adaptive mutations G4079A, G4944T and G6256A, resulting in NS2AR195K, NS3G122G, and NS3V560M, enhancing RNA replication. We demonstrated the utility of the POWV replicon system for high-throughput screening (HTS) assay to identify promising antivirals against POWV replication. We further explored the applications of the POWV replicon system, generating single-round infectious particles (SRIPs) by transfecting Huh7-POWV replicon cells with plasmids encoding viral capsid (C), premembrane (prM), and envelope (E) proteins, and revealed the distinct antigenic profiles of POWV with ZIKV. In summary, the POWV replicon and SRIP systems represent crucial platforms for genetic and functional analysis of the POWV life cycle and facilitating the discovery of antiviral drugs.IMPORTANCEIn light of the recent surge in human infections caused by POWV, a biosafety level-3 (BSL-3) classified virus, there is a pressing need to understand the viral life cycle and the development of effective countermeasures. To address this, we have pioneered the establishment of a POWV RNA replicon system and a replicon-based POWV SRIP system. Importantly, these systems are operable in BSL-2 laboratories, enabling comprehensive investigations into the viral life cycle and facilitating antiviral screening. In summary, these useful tools are poised to advance our understanding of the POWV life cycle and expedite the development of antiviral interventions.

Active Surveillance of Powassan Virus in Massachusetts Ixodes scapularis Ticks, Comparing Detection Using a New Triplex Real-Time PCR Assay with a Luminex Vector-Borne Panel.

Powassan virus is an emerging tick-borne pathogen capable of causing severe neuroinvasive disease. As the incidence of human Powassan virus grows both in magnitude and geographical range, the development of sensitive detection methods for diagnostics and surveillance is critical. In this study, a Taqman-based triplex real-time PCR assay was developed for the simultaneous and quantitative detection of Powassan virus and Powassan virus lineage II (deer tick virus) in Ixodes scapularis ticks. An exon-exon junction internal control was built-in to allow for accurate detection of RNA quality and the failure of RNA extraction. The newly developed assay was also applied to survey deer tick virus in tick populations at 13 sites on Cape Cod and Martha's Vineyard Island in Massachusetts. The assay's performance was compared with the Luminex xMAP MultiFLEX Vector-borne Panel 2. The results suggested that the real-time PCR method was more sensitive. Powassan virus infection rates among ticks collected from these highly endemic tick areas ranged from 0.0 to 10.4%, highlighting the fine-scale geographic variations in deer tick virus presence in this region. Looking forward, our PCR assay could be adopted in other Powassan virus surveillance systems.

The Vector Competence of Asian Longhorned Ticks in Langat Virus Transmission.

Haemaphysalis longicornis (the longhorned tick), the predominant tick species in China, serves as a vector for a variety of pathogens, and is capable of transmitting the tick-borne encephalitis virus (TBEV), the causative agent of tick-borne encephalitis. However, it is unclear how these ticks transmit TBEV. Langat virus (LGTV), which has a reduced pathogenicity in humans, has been used as a surrogate for TBEV. In this study, we aimed to investigate the vector competence of H. longicornis to transmit LGTV and demonstrate the efficient acquisition and transmission of LGTV between this tick species and mice. LGTV localization was detected in several tick tissues, such as the midgut, salivary glands, and synganglion, using quantitative PCR and immunohistochemical staining with a polyclonal antibody targeting the LGTV envelope protein. We demonstrated the horizontal transmission of LGTV to different developmental stages within the same generation but did not see evidence of vertical transmission. It was interesting to note that we observed mice acting as a bridge, facilitating the transmission of LGTV to neighboring naïve ticks during blood feeding. In conclusion, the virus-vector-host model employed in this study provides valuable insights into the replication and transmission of LGTV throughout its life cycle.

Proteomic and metabolomic analysis of the serum of patients with tick-borne encephalitis.

Tick-borne encephalitis virus (TBEV) is a common virus in Europe and Asia, causing around 10,000 to 10,500 infections annually. It affects the central nervous system and poses threats to public health. However, the exact molecular mechanisms of TBE pathogenesis are not yet fully understood due to the complex interactions between the virus and its host. In this study, a comprehensive analysis was conducted to characterize the serum metabolome and proteome of adult patients infected with TBEV, in comparison to a control group of healthy individuals. Liquid chromatography tandem mass spectrometry (LC-MS) was employed to monitor metabolic and proteomic alternations throughout the progression of the disease, significant physiological changes associated with different stages of the disease were identified. A total of 44 proteins and 115 metabolites exhibited significantly alternations in the sera of patients diagnosed with TBE. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of these metabolites and proteins revealed differential enrichment of genes associated with the extracellular matrix, complement binding, hemostasis, lipid metabolism, and amino acid metabolism between TBE patients and healthy controls. We gained valuable understanding of the specific metabolites implicated in the host's responses to TBE, establishing a basis for further research on TBE disease. SIGNIFICANCE: The current investigation revealed a comprehensive and systematic differences on TBE using LC-MS platform from human serum samples of TBE patients and healthy individuals providing the immune response to the invasion of TBE.

Molecular Detection and Phylogenetic Analysis of Tick-Borne Encephalitis Virus from Ticks Collected from Cattle in Kyrgyzstan, 2023.

Ticks are important vectors of the tick-borne encephalitis virus (TBEV). In Kyrgyzstan, the livestock farming trade and nomadic lifestyle enable tick-borne diseases to be imported from neighboring countries, but there are few relevant studies. In this study, we collected 40 ticks from cattle in Kyrgyzstan. Molecular marker analysis identified the ticks as Ixodes persulcatus (97.5%; n = 39) and Haemaphysalis punctata (2.5%; n = 1). Real-time PCR screening revealed two ticks to be positive for TBEV, but only one tick was amplified using nested PCR targeting the TBEV envelope (E) and non-structure 5 (NS5) gene. The obtained sequences belonged to the TBEV Siberian subtype and phylogenetic tree analysis results confirmed that the virus was related to the Bosnia strain. We also performed next-generation sequencing, which confirmed the TBEV Siberian subtype. Continuous research and surveillance of TBEV in Kyrgyzstan are required to provide further information on tick-borne diseases.

Structural diversity of tick-borne encephalitis virus particles in the inactivated vaccine based on strain Sofjin.

The main approach to preventing tick-borne encephalitis (TBE) is vaccination. Formaldehyde-inactivated TBE vaccines have a proven record of safety and efficiency but have never been characterized structurally with atomic resolution. We report a cryoelectron microscopy (cryo-EM) structure of the formaldehyde-inactivated TBE virus (TBEV) of Sofjin-Chumakov strain representing the Far-Eastern subtype. A 3.8 Å resolution reconstruction reveals the structural integrity of the envelope E proteins, specifically the E protein ectodomains. The comparative study shows a high structural similarity to the previously published structures of the TBEV European subtype strains Hypr and Kuutsalo-14. A fraction of inactivated virions exhibits asymmetric features including the deformations of the membrane profile. We propose that the heterogeneity is caused by inactivation and perform a local variability analysis on the small parts of the envelope protein shell to reveal membrane curvature features possibly induced by the inactivation. The results of this study will have implications for the design of novel vaccines against diseases caused by flaviviruses.

Antiviral activity of porphyrins and porphyrin-like compounds against tick-borne encephalitis virus: Blockage of the viral entry/fusion machinery by photosensitization-mediated destruction of the viral envelope.

Tick-borne encephalitis virus (TBEV), the causative agent of tick-borne encephalitis (TBE), is a medically important flavivirus endemic to the European-Asian continent. Although more than 12,000 clinical cases are reported annually worldwide, there is no anti-TBEV therapy available to treat patients with TBE. Porphyrins are macrocyclic molecules consisting of a planar tetrapyrrolic ring that can coordinate a metal cation. In this study, we investigated the cytotoxicity and anti-TBEV activity of a large series of alkyl- or (het)aryl-substituted porphyrins, metalloporphyrins, and chlorins and characterized their molecular interactions with the viral envelope in detail. Our structure-activity relationship study showed that the tetrapyrrole ring is an essential structural element for anti-TBEV activity, but that the presence of different structurally distinct side chains with different lengths, charges, and rigidity or metal cation coordination can significantly alter the antiviral potency of porphyrin scaffolds. Porphyrins were demonstrated to interact with the TBEV lipid membrane and envelope protein E, disrupt the TBEV envelope and inhibit the TBEV entry/fusion machinery. The crucial mechanism of the anti-TBEV activity of porphyrins is based on photosensitization and the formation of highly reactive singlet oxygen. In addition to blocking viral entry and fusion, porphyrins were also observed to interact with RNA oligonucleotides derived from TBEV genomic RNA, indicating that these compounds could target multiple viral/cellular structures. Furthermore, immunization of mice with porphyrin-inactivated TBEV resulted in the formation of TBEV-neutralizing antibodies and protected the mice from TBEV infection. Porphyrins can thus be used to inactivate TBEV while retaining the immunogenic properties of the virus and could be useful for producing new inactivated TBEV vaccines.

Kyasanur Forest disease virus NS3 helicase: Insights into structure, activity, and inhibitors.

Kyasanur Forest disease virus (KFDV), a tick-borne flavivirus prevalent in India, presents a serious threat to human health. KFDV NS3 helicase (NS3hel) is considered a potential drug target due to its involvement in the viral replication complex. Here, we resolved the crystal structures of KFDV NS3hel apo and its complex with three phosphate molecules, which indicates a conformational switch during ATP hydrolysis. Our data revealed that KFDV NS3hel has a higher binding affinity for dsRNA, and its intrinsic ATPase activity was enhanced by dsRNA while being inhibited by DNA. Through mutagenesis analysis, several residues within motifs I, Ia, III, V, and VI were identified to be crucial for NS3hel ATPase activity. Notably, the M419A mutation drastically reduced NS3hel ATPase activity. We propose that the methionine-aromatic interaction between residues M419 and W294, located on the surface of the RNA-binding channel, could be a target for the design of efficient inhibitor probes. Moreover, epigallocatechin gallate (EGCG), a tea-derived polyphenol, strongly inhibited NS3hel ATPase activity with an IC50 value of 0.8 µM. Our computational docking data show that EGCG binds at the predicted druggable hotspots of NS3hel. Overall, these findings contribute to the development and design of more effective and specific inhibitors.