Nine-year seroepidemiological study of severe fever with thrombocytopenia syndrome virus infection in feral horses in Cape Toi, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is a fatal zoonosis caused by ticks in East Asia. As SFTS virus (SFTSV) is maintained between wildlife and ticks, seroepidemiological studies in wildlife are important to understand the behavior of SFTSV in the environment. Miyazaki Prefecture, Japan, is an SFTS-endemic area, and approximately 100 feral horses, called Misaki horses (Equus caballus), inhabit Cape Toi in Miyazaki Prefecture. While these animals are managed in a wild-like manner, their ages are ascertainable due to individual identification. In the present study, we conducted a seroepidemiological survey of SFTSV in Misaki horses between 2015 and 2023. This study aimed to understand SFTSV infection in horses and its transmission to wildlife. A total of 707 samples from 180 feral horses were used to determine the seroprevalence of SFTSV using enzyme-linked immunosorbent assay (ELISA). Neutralization testing was performed on 118 samples. In addition, SFTS viral RNA was detected in ticks from Cape Toi and feral horses. The overall seroprevalence between 2015 and 2023 was 78.5% (555/707). The lowest seroprevalence was 55% (44/80) in 2016 and the highest was 92% (76/83) in 2018. Seroprevalence was significantly affected by age, with 11% (8/71) in those less than one year of age and 96.7% (435/450) in those four years of age and older (p < 0.0001). The concordance between ELISA and neutralization test results was 88.9% (105/118). SFTS viral RNA was not detected in ticks (n = 516) or feral horses. This study demonstrated that horses can be infected with SFTSV and that age is a significant factor in seroprevalence in wildlife. This study provides insights into SFTSV infection not only in horses but also in wildlife in SFTS-endemic areas.

Seromolecular survey and risk factor analysis of Crimean-Congo haemorrhagic fever orthonairovirus in occupationally exposed herdsmen and unexposed febrile patients in Kwara State, Nigeria.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a globally significant tick-borne zoonotic pathogen that causes fatal haemorrhagic disease in humans. Despite constituting an ongoing public health threat, limited research exists on the presence of CCHFV among herdsmen, an occupationally exposed population that has prolonged contact with ruminants and ticks. This cross-sectional study, conducted between October 2018 and February 2020 in Kwara State, Nigeria, was aimed at assessing CCHFV seroprevalence among herdsmen and non-herdsmen febrile patients, and identifying the associated risk factors. Blood samples from herdsmen (n = 91) and febrile patients in hospitals (n = 646) were analyzed for anti-CCHFV IgG antibodies and CCHFV S-segment RNA using ELISA and RT-PCR, respectively. Results revealed a remarkably high CCHFV seroprevalence of 92.3% (84/91) among herdsmen compared to 7.1% (46/646) in febrile patients. Occupational risk factors like animal and tick contact, tick bites, and hand crushing of ticks significantly contributed to higher seroprevalence in the herdsmen (p<0.0001). Herdsmen were 156.5 times more likely (p<0.0001) to be exposed to CCHFV than febrile patients. Notably, the odds of exposure were significantly higher (OR = 191.3; p<0.0001) in herdsmen with a history of tick bites. Although CCHFV genome was not detectable in the tested sera, our findings reveal that the virus is endemic among herdsmen in Kwara State, Nigeria. CCHFV should be considered as a probable cause of febrile illness among humans in the study area. Given the nomadic lifestyle of herdsmen, further investigations into CCHF epidemiology in this neglected population are crucial. This study enhances our understanding of CCHFV dynamics and emphasizes the need for targeted interventions in at-risk communities.

Tick-borne diseases in Europe: Current prevention, control tools and the promise of aptamers.

In Europe, tick-borne diseases (TBDs) cause significant morbidity and mortality, affecting both human and animal health. Ticks can transmit a wide variety of pathogens (bacteria, viruses, and parasites) and feed on many vertebrate hosts. The incidence and public health burden of TBDs are tending to intensify in Europe due to various factors, mainly anthropogenic and often combined. Early detection of tick-borne pathogens (TBPs), preventive measures and treatment are of great importance to control TBDs and their expansion. However, there are various limitations in terms of the sensitivity and/or specificity of detection and prevention methods, and even in terms of feasibility. Aptamers are single-stranded DNA or RNA that could address these issues as they are able to bind with high affinity and specificity to a wide range of targets (e.g., proteins, small compounds, and cells) due to their unique three-dimensional structure. To date, aptamers have been selected against TBPs such as tick-borne encephalitis virus, Francisella tularensis, and Rickettsia typhi. These studies have demonstrated the benefits of aptamer-based assays for pathogen detection and medical diagnosis. In this review, we address the applications of aptamers to TBDs and discuss their potential for improving prevention measures (use of chemical acaricides, vaccination), diagnosis and therapeutic strategies to control TBDs.

Genetic background of adaptation of Crimean-Congo haemorrhagic fever virus to the different tick hosts.

Crimean-Congo haemorrhagic fever orthonairovirus (CCHFV) is a negative-sense, single-stranded RNA virus with a segmented genome and the causative agent of a severe Crimean-Congo haemorrhagic fever (CCHF) disease. The virus is transmitted mainly by tick species in Hyalomma genus but other ticks such as representatives of genera Dermacentor and Rhipicephalus may also be involved in virus life cycle. To improve our understanding of CCHFV adaptation to its tick species, we compared nucleotide composition and codon usage patterns among the all CCHFV strains i) which sequences and other metadata as locality of collection and date of isolation are available in GenBank and ii) which were isolated from in-field collected tick species. These criteria fulfilled 70 sequences (24 coding for S, 23 for M, and 23 for L segment) of virus isolates originating from different representatives of Hyalomma and Rhipicephalus genera. Phylogenetic analyses confirmed that Hyalomma- and Rhipicephalus-originating CCHFV isolates belong to phylogenetically distinct CCHFV clades. Analyses of nucleotide composition among the Hyalomma- and Rhipicephalus-originating CCHFV isolates also showed significant differences, mainly in nucleotides located at the 3rd codon positions indicating changes in codon usage among these lineages. Analyses of codon adaptation index (CAI), effective number of codons (ENC), and other codon usage statistics revealed significant differences between Hyalomma- and Rhipicephalus-isolated CCHFV strains. Despite both sets of strains displayed a higher adaptation to use codons that are preferred by Hyalomma ticks than Rhipicephalus ticks, there were distinct codon usage preferences observed between the two tick species. These findings suggest that over the course of its long co-evolution with tick vectors, CCHFV has optimized its codon usage to efficiently utilize translational resources of Hyalomma species.

Crimean-Congo Hemorrhagic Fever Virus in Ticks Collected from Cattle, Corsica, France, 2023.

We report the detection of Crimean-Congo hemorrhagic fever virus (CCHFV) in Corsica, France. We identified CCHFV African genotype I in ticks collected from cattle at 2 different sites in southeastern and central-western Corsica, indicating an established CCHFV circulation. Healthcare professionals and at-risk groups should be alerted to CCHFV circulation in Corsica.

Identification and epidemiological study of an uncultured flavivirus from ticks using viral metagenomics and pseudoinfectious viral particles.

During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.

Crimean-Congo Hemorrhagic Fever Virus for Clinicians-Epidemiology, Clinical Manifestations, and Prevention.

Crimean-Congo hemorrhagic fever (CCHF) is a tickborne infection that can range from asymptomatic to fatal and has been described in >30 countries. Early identification and isolation of patients with suspected or confirmed CCHF and the use of appropriate prevention and control measures are essential for preventing human-to-human transmission. Here, we provide an overview of the epidemiology, clinical features, and prevention and control of CCHF. CCHF poses a continued public health threat given its wide geographic distribution, potential to spread to new regions, propensity for genetic variability, and potential for severe and fatal illness, in addition to the limited medical countermeasures for prophylaxis and treatment. A high index of suspicion, comprehensive travel and epidemiologic history, and clinical evaluation are essential for prompt diagnosis. Infection control measures can be effective in reducing the risk for transmission but require correct and consistent application.

Crimean Congo Hemorrhagic Fever Virus for Clinicians-Virology, Pathogenesis, and Pathology.

Crimean-Congo hemorrhagic fever (CCHF), caused by CCHF virus, is a tickborne disease that can cause a range of illness outcomes, from asymptomatic infection to fatal viral hemorrhagic fever; the disease has been described in >30 countries. We conducted a literature review to provide an overview of the virology, pathogenesis, and pathology of CCHF for clinicians. The virus life cycle and molecular interactions are complex and not fully described. Although pathogenesis and immunobiology are not yet fully understood, it is clear that multiple processes contribute to viral entry, replication, and pathological damage. Limited autopsy reports describe multiorgan involvement with extravasation and hemorrhages. Advanced understanding of CCHF virus pathogenesis and immunology will improve patient care and accelerate the development of medical countermeasures for CCHF.

Viromes of Haemaphysalis longicornis reveal different viral abundance and diversity in free and engorged ticks.

Haemaphysalis longicornis ticks, commonly found in East Asia, can transmit various pathogenic viruses, including the severe fever with thrombocytopenia syndrome virus (SFTSV) that has caused febrile diseases among humans in Hubei Province. However, understanding of the viromes of H. longicornis was limited, and the prevalence of viruses among H. longicornis ticks in Hubei was not well clarified. This study investigates the viromes of both engorged (fed) and free (unfed) H. longicornis ticks across three mountainous regions in Hubei Province from 2019 to 2020. RNA-sequencing analysis identified viral sequences that were related to 39 reference viruses belonging to unclassified viruses and seven RNA viral families, namely Chuviridae, Nairoviridae, Orthomyxoviridae, Parvoviridae, Phenuiviridae, Rhabdoviridae, and Totiviridae. Viral abundance and diversity in these ticks were analysed, and phylogenetic characteristics of the Henan tick virus (HNTV), Dabieshan tick virus (DBSTV), Okutama tick virus (OKTV), and Jingmen tick virus (JMTV) were elucidated based on their full genomic sequences. Prevalence analysis demonstrated that DBSTV was the most common virus found in individual H. longicornis ticks (12.59%), followed by HNTV (0.35%), whereas JMTV and OKTV were not detected. These results improve our understanding of H. longicornis tick viromes in central China and highlight the role of tick feeding status and geography in shaping the viral community. The findings of new viral strains and their potential impact on public health raise the need to strengthen surveillance efforts for comprehensively assessing their spillover potentials.

Screening and identification of persistent viruses in cell lines derived from medically important arthropods.

Arthropod-derived cell lines serve as crucial tools for studying arthropod-borne viruses (arboviruses). However, it has recently come to light that certain cell lines harbor persistent infections of arthropod-specific viruses, which do not cause any apparent cytopathic effects. Moreover, some of these persistent viral infections either inhibit or promote the growth of arboviruses. Therefore, it is of utmost importance to identify the presence of such persistent viruses and understand their impact on arboviral infections. In this study, we conducted a comprehensive virome analysis of several arthropod-derived cell lines, including mosquito-derived NIID-CTR, Ar-3, MSQ43, NIAS-AeAl-2, CCL-126 cells, and tick-derived IDE8 cells, along with flesh fly-derived NIH-Sape-4 cells. The aim was to determine if these cells were infected with persistent viruses. The results revealed the presence of 15 persistent viruses in NIID-CTR, Ar-3, MSQ43, NIAS-AeAl-2, and IDE8 cells. Among these, 11 were already known arthropod-specific viruses, while the remaining 4 were novel viruses belonging to Orthophasmavirus, Rhabdoviridae, Totiviridae, and Bunyavirales. In contrast, CCL-126 and NIH-Sape-4 cells appeared to be free of viral infections. This study provides valuable insights into the diversity and latency of arthropod-specific viruses within arthropod-derived cell lines. Further investigations are required to explore persistent viral infections in other arthropod-derived cell cultures and their effects on arbovirus replication. Understanding these factors will enhance the accuracy and reliability of experimental data obtained using these cell lines.

Epidemiological and Molecular Study on Tick-Borne Pathogens in Argun Port Area Near the Chinese-Russian Border.

Objective: We aim to investigate the species composition of ticks and the pathogen characteristics they carry in the Argun port area of the China-Russia border. Materials and Methods: Ticks were collected in surrounding grassland, mixed forest land, and other different habitats around the Argun port area at the Sino-Russian Border of Inner Mongolia in China in April 2019. The presence of 16 potential pathogens, including Yersinia Pestis, Francisella tularensis, Coxiella burnetii (Cb), Anaplasma sp. (Ap), spotted fever group rickettsiae (SFG Rk), Borrelia sp. (Bl), Leptospira, Bartonella spp., Babesia, Crimean-Congo hemorrhagic fever virus, tick-borne encephalitis virus, Bhanja virus, West Nile Virus, severe fever with thrombocytopenia syndrome bunyavirus, Hantaan virus, and bocavirus (boca) was analyzed by polymerase chain reaction. The DNA and amino acid sequences of tick-borne pathogens were compared for homology, and the phylogenetic trees were constructed by using Mega and Lasergene software. Results: A total of 210 ticks were collected and they belonged to three species: Dermacentor nuttalli, Ixodes persulcatus, and Haemaphysalis verticalis. Among them, 165 (78.57%) ticks tested positive for 5 pathogens, namely Ap, SFG Rk, Cb, Bl, and boca. Fifteen (7.14%) ticks were detected coinfection with two pathogens, and none were coinfected with three or more pathogens. Conclusion: This study shows the prevalence of at least five tick-borne pathogens in Argun, and there is a risk of coinfection by two pathogens in one tick. This study reveals the great importance of controlling tick-borne diseases in this region.

The tick saliva peptide HIDfsin2 promotes the tick-borne virus SFTSV replication in vitro by enhancing p38 signal pathway.

Pathogens co-evolved with ticks to facilitate blood collection and pathogen transmission. Although tick saliva was recently found to be rich in bioactive peptides, it is still elusive which saliva peptide promotes virus transmission and which pathways are invovled. Here, we used a saliva peptide HIDfsin2 and a severe fever with thrombocytopenia syndrome virus (SFTSV) both carried by the tick Haemaphysalis longicornis to elucidate the relationship between tick saliva components and tick-borne viruses. HIDfsin2 was found to promote the replication of SFTSV in a dose-dependent manner in vitro. HIDfsin2 was further revealed to MKK3/6-dependently magnify the activation of p38 MAPK. The overexpression, knockdown and phosphorylation site mutation of p38α indicated that p38 MAPK activation facilitated SFTSV infection in A549 cells. Moreover, the blockade of p38 MAPK activation significantly suppressed SFTSV replication. Differently, HIDfsin2 or pharmacological inhibition of p38 MAPK activation had no effect on a mosquito-borne Zika virus (ZIKV). All these results showed that HIDfsin2 specifically promoted SFTSV replication through the MKK3/6-dependent enhancement of p38 MAPK activation. Our study provides a new perspective on the transmission of tick-borne viruses under natural conditions, and supports that the blockade of p38 MAPK activation can be a promising strategy against the mortal tick-borne virus SFTSV.

Ticks infected with Crimean-Congo hemorrhagic fever virus (CCHFV): A decision approach systematic review and meta-analysis regarding their role as vectors.

BACKGROUND: There are many studies present in literature performed to isolate CCHFV from ticks. However, gaps in knowledge for estimating global annual CCHFV infection rates; global CCHFV infection rates and records; CCHFV infection trend in ticks and ticks infested animals; and lack a decision regarding the role of ticks as CCHFV vectors caused to conduct this review. METHODS: From 605 papers identified, 150 papers were selected to become the study meta-analysis (57 papers), detailed CCHFV infected ticks (63 papers), and systematic review (99 papers) parts. RESULTS: Since there are no significant differences between the annual tick CCHFV infection rates; tick CCHFV infection rates between continents; and CCHFV infection rates between tick species and gender of ticks, and ticks and tick species infested animals. Given that ticks are considered both as CCHFV reservoirs and vectors. It is possible a little burden of CCHFV in infected ticks in natural enzootic cycles to maintain and disseminate CCHFV in humans. The decision regarding the role of ticks as CCHFV vectors administrated based on the study and a separately literature search regarding the role of ticks as CCHFV vectors; CCHFV infection rates and records; and trend of CCHFV infection records in 31 tick species. The trend of CCHFV infection records in 31 tick species exhibited a decreasing trend indicating the degree and importance of their roles as CCHFV vectors. CONCLUSIONS: Among 31 CCHFV infected tick species, 15 species have been enrolled as proven vectors and 16 species are suspected as potential vectors.

Recent Progress on Tick-Borne Animal Diseases of Veterinary and Public Health Significance in China.

Ticks and tick-borne diseases pose a growing threat to human and animal health, which has brought great losses to livestock production. With the continuous expansion of human activities and the development of natural resources, there are more and more opportunities for humans to contract ticks and tick-borne pathogens. Therefore, research on ticks and tick-borne diseases is of great significance. This paper reviews recent progress on tick-borne bacterial diseases, viral diseases, and parasitic diseases in China, which provides a theoretical foundation for the research of tick-borne diseases.

Spatial patterns of West Nile virus distribution in the Volgograd region of Russia, a territory with long-existing foci.

Southern Russia remains affected by West Nile virus (WNV). In the current study, we identified the spatial determinants of WNV distribution in an area with endemic virus transmission, with special reference to the urban settings, by mapping probable points of human infection acquisition and points of virus detection in mosquitoes, ticks, birds, and mammals during 1999-2016. The suitability of thermal conditions for extrinsic virus replication was assessed based on the approach of degree-day summation and their changes were estimated by linear trend analysis. A generalized linear model was used to analyze the year-to-year variation of human cases versus thermal conditions. Environmental suitability was determined by ecological niche modelling using MaxEnt software. Human population density was used as an offset to correct for possible bias. Spatial analysis of virus detection in the environment showed significant contributions from surface temperature, altitude, and distance from water bodies. When indicators of location and mobility of the human population were included, the relative impact of factors changed, with roads becoming most important. When the points of probable human case infection were added, the percentage of leading factors changed only slightly. The urban environment significantly increased the epidemic potential of the territory and created quite favorable conditions for virus circulation. The private building sector with low-storey houses and garden plots located in the suburbs provided a connection between urban and rural transmission cycles.

Epidemiology of West Nile virus in Africa: An underestimated threat.

BACKGROUND: West Nile virus is a mosquito-borne flavivirus which has been posing continuous challenges to public health worldwide due to the identification of new lineages and clades and its ability to invade and establish in an increasing number of countries. Its current distribution, genetic variability, ecology, and epidemiological pattern in the African continent are only partially known despite the general consensus on the urgency to obtain such information for quantifying the actual disease burden in Africa other than to predict future threats at global scale. METHODOLOGY AND PRINCIPAL FINDINGS: References were searched in PubMed and Google Scholar electronic databases on January 21, 2020, using selected keywords, without language and date restriction. Additional manual searches of reference list were carried out. Further references have been later added accordingly to experts' opinion. We included 153 scientific papers published between 1940 and 2021. This review highlights: (i) the co-circulation of WNV-lineages 1, 2, and 8 in the African continent; (ii) the presence of diverse WNV competent vectors in Africa, mainly belonging to the Culex genus; (iii) the lack of vector competence studies for several other mosquito species found naturally infected with WNV in Africa; (iv) the need of more competence studies to be addressed on ticks; (iv) evidence of circulation of WNV among humans, animals and vectors in at least 28 Countries; (v) the lack of knowledge on the epidemiological situation of WNV for 19 Countries and (vii) the importance of carrying out specific serological surveys in order to avoid possible bias on WNV circulation in Africa. CONCLUSIONS: This study provides the state of art on WNV investigation carried out in Africa, highlighting several knowledge gaps regarding i) the current WNV distribution and genetic diversity, ii) its ecology and transmission chains including the role of different arthropods and vertebrate species as competent reservoirs, and iii) the real disease burden for humans and animals. This review highlights the needs for further research and coordinated surveillance efforts on WNV in Africa.

Comparative Study of Ten Thogotovirus Isolates and Their Distinct In Vivo Characteristics.

Thogotoviruses are tick-borne arboviruses that comprise a unique genus within the Orthomyxoviridae family. Infections with thogotoviruses primarily cause disease in livestock with occasional reports of human infections suggesting a zoonotic potential. In the past, multiple genetically distinct thogotoviruses were isolated mostly from collected ticks. However, many aspects regarding their phylogenetic relationships, morphological characteristics, and virulence in mammals remain unclear. For the present comparative study, we used a collection of 10 different thogotovirus isolates from different geographic areas. Next-generation sequencing and subsequent phylogenetic analyses revealed a distinct separation of these viruses into two major clades, the Thogoto-like and Dhori-like viruses. Electron microscopy demonstrated a heterogeneous morphology with spherical and filamentous particles being present in virus preparations. To study their pathogenicity, we analyzed the viruses in a small animal model system. In intraperitoneally infected C57BL/6 mice, all isolates showed a tropism for liver, lung, and spleen. Importantly, we did not observe horizontal transmission to uninfected, highly susceptible contact mice. The isolates enormously differed in their capacity to induce disease, ranging from subclinical to fatal outcomes. In vivo multistep passaging experiments of two low-pathogenic isolates showed no increased virulence and sequence analyses of the passaged viruses indicated a high stability of the viral genomes after 10 mouse passages. In summary, our analysis demonstrates the broad genetic and phenotypic variability within the thogotovirus genus. Moreover, thogotoviruses are well adapted to mammals but their horizontal transmission seems to depend on ticks as their vectors. IMPORTANCE Since their discovery over 60 years ago, 15 genetically distinct members of the thogotovirus genus have been isolated. These arboviruses belong to the Orthomyxovirus family and share many features with influenza viruses. However, numerous of these isolates have not been characterized in depth. In the present study, we comparatively analyzed a collection of 10 different thogotovirus isolates to answer basic questions about their phylogenetic relationships, morphology, and pathogenicity in mice. Our results highlight shared and unique characteristics of this diverse genus. Taken together, these observations provide a framework for the phylogenic classification and phenotypic characterization of newly identified thogotovirus isolates that could potentially cause severe human infections as exemplified by the recently reported, fatal Bourbon virus cases in the United States.

Epidemiological study of Kabuto Mountain virus, a novel uukuvirus, in Japan.

Kabuto Mountain virus (KAMV), the new member of the genus Uukuvirus, was isolated from the tick Haemaphysalis flava in 2018 in Japan. To date, there is no information on KAMV infection in human and animals. Therefore, serological surveillance of the infection among humans and wild mammals was conducted by virus-neutralization (VN) test and indirect immunofluorescence assay (IFA). Sera of 24 humans, 59 monkeys, 171 wild boars, 233 Sika deer, 7 bears, and 27 nutria in Yamaguchi Prefecture were analyzed by VN test. The positive ratio of humans, monkeys, wild boars, and Sika deer were 20.8%, 3.4%, 33.9% and 4.7%, respectively. No positive samples were detected in bears and nutria. The correlation coefficients between VN test and IFA in human, monkey, wild boar, and Sika deer sera were 0.5745, 0.7198, 0.9967 and 0.9525, respectively. In addition, KAMV was detected in one pool of Haemaphysalis formosensis ticks in Wakayama Prefecture. These results indicated that KAMV or KAMV-like virus is circulating among many wildlife and ticks, and that this virus incidentally infects humans.

Roles of ESCRT Proteins ALIX and CHMP4A and Their Interplay with Interferon-Stimulated Gene 15 during Tick-Borne Flavivirus Infection.

Flaviviruses are usually transmitted to humans via mosquito or tick bites. During infection, virus replication and assembly, whose cellular sites are relatively close, are controlled by virus proteins and a diverse range of host proteins. By siRNA-mediated gene silencing, we showed that ALIX and CHMP4A, two members of the host endosomal sorting complex required for transport (ESCRT) protein machinery, are required during flavivirus infection. Using cell lines expressing subgenomic replicons and replicon virus-like particles, we demonstrated specific roles for ALIX and CHMP4A in viral replication and assembly, respectively. Employing biochemical and imaging methodology, we showed that the ESCRT proteins are recruited by a putative specific late (L) domain motif LYXLA within the NS3 protein of tick-borne flaviviruses. Furthermore, to counteract the recruitment of ESCRT proteins, the host cells may elicit defense mechanisms. We found that ectopic expression of the interferon-stimulated gene 15 (ISG15) or the E3 ISG15-protein ligase (HERC5) reduced virus replication by suppressing the positive effects of ALIX and CHMP4A. Collectively, these results have provided new insights into flavivirus-host cell interactions that function as checkpoints, including the NS3 and the ESCRT proteins, the ISG15 and the ESCRT proteins, at essential stages of the virus life cycle. IMPORTANCE Flaviviruses are important zoonotic viruses with high fatality rates worldwide. Here, we report that during infection, the virus employs members of ESCRT proteins for virus replication and assembly. Among the ESCRT proteins, ALIX acts during virus replication, while CHMP4A is required during virus assembly. Another important ESCRT protein, TSG101, is not required for virus production. The ESCRT, complex, ALIX-CHMP4A, is recruited to NS3 through their interactions with the putative L domain motif of NS3, while CHMP4A is recruited to E. In addition, we demonstrate the antiviral mechanism of ISG15 and HERC5, which degrades ALIX and CHIMP4A, indirectly targets virus infection. In summary, we reveal host-dependency factors supporting flavivirus infection, but these factors may also be targeted by antiviral host effector mechanisms.