Hazara virus and Crimean-Congo Hemorrhagic Fever Virus show a different pattern of entry in fully-polarized Caco-2 cell line.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) and Hazara virus (HAZV) belong to the same viral serotype and family. HAZV has lately been used as a model system and surrogate to CCHFV. However, virus-host cell interaction and level of pathogenicity for these viruses are not well investigated nor compared. In this study, we compared HAZV and CCHFV infection of human polarized epithelial cells to shed light on similarities and differences in virus-host cell interaction between these two viruses. We investigated the pattern of infection of CCHFV and HAZV in fully polarized human cells, the Caco-2 cell line. Polarization of Caco-2 cells lead to difference in expression level and pattern of proteins between the apical and the basolateral membranes. We found that CCHFV virus, in contrast to HAZV, is more likely infecting polarized cells basolaterally. In addition, we found that cytokines/pro-inflammatory factors or other viral factors secreted from CCHFV infected moDC cells enhance the entry of CCHFV contrary to HAZV. We have shown that CCHFV and HAZV early in infection use different strategies for entry. The data presented in this study also highlight the important role of cytokines in CCHFV-host cell interaction.

Differential Growth Characteristics of Crimean-Congo Hemorrhagic Fever Virus in Kidney Cells of Human and Bovine Origin.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes a lethal tick-borne zoonotic disease with severe clinical manifestation in humans but does not produce symptomatic disease in wild or domestic animals. The factors contributing to differential outcomes of infection between species are not yet understood. Since CCHFV is known to have tropism to kidney tissue and cattle play an important role as an amplifying host for CCHFV, in this study, we assessed in vitro cell susceptibility to CCHFV infection in immortalized and primary kidney and adrenal gland cell lines of human and bovine origin. Based on our indirect fluorescent focus assay (IFFA), we suggest a cell-to-cell CCHF viral spread process in bovine kidney cells but not in human cells. Over the course of seven days post-infection (dpi), infected bovine kidney cells are found in restricted islet-like areas. In contrast, three dpi infected human kidney or adrenal cells were noted in areas distant from one another yet progressed to up to 100% infection of the monolayer. Pronounced CCHFV replication, measured by quantitative real-time RT-PCR (qRT-PCR) of both intra- and extracellular viral RNA, was documented only in human kidney cells, supporting restrictive infection in cells of bovine origin. To further investigate the differences, lactate dehydrogenase activity and cytopathic effects were measured at different time points in all mentioned cells. In vitro assays indicated that CCHFV infection affects human and bovine kidney cells differently, where human cell lines seem to be markedly permissive. This is the initial reporting of CCHFV susceptibility and replication patterns in bovine cells and the first report to compare human and animal cell permissiveness in vitro. Further investigations will help to understand the impact of different cell types of various origins on the virus-host interaction.

Favipiravir (T-705) but not ribavirin is effective against two distinct strains of Crimean-Congo hemorrhagic fever virus in mice.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a cause of serious hemorrhagic disease in humans. Humans infected with CCHFV develop a non-specific febrile illness and then progress to the hemorrhagic phase where case fatality rates can be as high as 30%. Currently there is lack of vaccines and the recommended antiviral treatment, ribavirin, has inconsistent efficacy in both human and animal studies. In this study we developed a model of CCHFV infection in type I interferon deficient mice using the clinical CCHFV isolate strain Hoti. Mice infected with strain Hoti develop a progressively worsening and ultimately fatal disease. We utilized this model along with our established model using the prototypical CCHFV strain 10200 to evaluate treatment with ribavirin or the antiviral favipiravir. While ribavirin treatment was able to suppress viral loads at early time points it was ultimately unable to prevent development of terminal disease in mice infected with either strain of CCHFV. In contrast, favipiravir showed clinical benefit even when administered late in the clinical progression of CCHF. Interestingly, in a small subset of mice, late-onset of CCHF was observed after favipiravir treatment was stopped and persistence of viral RNA in favipiravir treated survivors was also seen. Nevertheless, favipiravir showed significant clinical benefit against two distinct strains of CCHFV suggesting it may be a potent antiviral for treatment of human CCHFV infections.

Susceptibility of tick cell lines to infection with Alkhumra haemorrhagic fever virus.

BACKGROUND: Although Alkhumra haemorrhagic fever virus (AHFV) has been isolated from ticks, epidemiological data suggest that it is transmitted from livestock to humans by direct contact with animals or by mosquito bites, but not by ticks. This study was carried out to assess the ability of the virus to replicate in tick cells in vitro. METHODS: AHFV was inoculated into cell lines derived from the hard ticks Hyalomma anatolicum (HAE/CTVM9) and Rhipicephalus appendiculatus (RAE/CTVM1) and the soft tick Ornithodoros moubata (OME/CTVM24). Inoculated cells were directly examined every week for 4 weeks by real-time reverse transcription PCR and by IFAT using polyclonal antibodies. RESULTS: AHFV RNA was detected in all three inoculated tick cell lines throughout the 4-week observation period at levels up to almost twice that of the inoculum, but none of them exhibited a cytopathic effect. AHFV antigen could be detected in all three cell lines by IFAT. Titration of tick cell culture suspension in LLC-MK2 cells yielded AHFV titres of 10(6.6) 50% tissue culture infective dose (TCID50)/ml for OME/CTVM24 and 10(5.5) TCID50/ml for RAE/CTVM1 cells after 4 weeks of culturing; no viable virus was detected in HAE/CTVM9 cells. CONCLUSION: This is the first description of propagation of AHFV in tick cells.

Influence of laboratory animal hosts on the life cycle of Hyalomma marginatum and implications for an in vivo transmission model for Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the most geographically widespread arboviruses and causes a severe hemorrhagic syndrome in humans. The virus circulates in nature in a vertebrate-tick cycle and ticks of the genus Hyalomma are the main vectors and reservoirs. Although the tick vector plays a central role in the maintenance and transmission of CCHFV in nature, comparatively little is known of CCHFV-tick interactions. This is mostly due to the fact that establishing tick colonies is laborious, and working with CCHFV requires a biosafety level 4 laboratory (BSL4) in many countries. Nonetheless, an in vivo transmission model is essential to understand the epidemiology of the transmission cycle of CCHFV. In addition, important parameters such as vectorial capacity of tick species, levels of infection in the host necessary to infect the tick, and aspects of virus transmission by tick bite including the influence of tick saliva, cannot be investigated any other way. Here, we evaluate the influence of different laboratory animal species as hosts supporting the life cycle of Hyalomma marginatum, a two-host tick. Rabbits were considered the host of choice for the maintenance of the uninfected colonies due to high larval attachment rates, shorter larval-nymphal feeding times, higher nymphal molting rates, high egg hatching rates, and higher conversion efficiency index (CEI). Furthermore, we describe the successful establishment of an in vivo transmission model for CCHFV in a BSL4 biocontainment setting using interferon knockout mice. This will give us a new tool to study the transmission and interaction of CCHFV with its tick vector.

Infection and propagation of Crimean-Congo hemorrhagic fever virus in embryonated chicken eggs.

The embryonated chicken egg (ECE) provides a convenient, space-saving incubator for the cultivation of many kinds of animal viruses where the egg can be easily observed for viral replication throughout the development of the chicken embryo. Within the family Bunyaviridae, the embryonated egg has been used as a host system for many viruses such as Rift Valley fever virus and Akabane virus. The current study was conducted to determine the cultivation of Crimean-Congo hemorrhagic fever virus (CCHFV) in ECE. Four-day-old eggs were infected with CCHFV via the yolk sac route and harvested embryonic tissues and amino-allantoic fluid (AAF) that were used for virus passage and viral RNA (vRNA) detection. Quantification of vRNA copies was performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Our study indicated that CCHFV caused the death of the embryonated egg in a dose-dependent manner and the 50% egg infectious dose (EID50) was determined to be 6.47×10(5) copies/egg. CCHFV replicated and passaged well in the egg and high viral loads were detected both in embryonic tissue (10(9-10) copies/g) and AAF (10(7-9) copies/ml) of the embryonated egg. Thus, ECE could be used for viral cultivation and preservation, and as a potential host infection model for the study of the pathogenesis of CCHFV.

Factors driving the circulation and possible expansion of Crimean-Congo haemorrhagic fever virus in the western Palearctic.

AIMS: To produce a spatial risk map regarding spread of Crimean-Congo haemorrhagic fever virus (CCHFV) in the western Palearctic by linking a process-driven model of the main tick vector, Hyalomma marginatum, to a Next Generation Matrix. METHODS AND RESULTS: Process-driven model was composed of deterministic equations that simulate developmental and mortality rates of different tick stages by using temperature and atmospheric water vapour data. The model used climate data at 10-day intervals at a spatial resolution of 10 min over western Palearctic. The model estimated the basic reproduction number, R0, for CCHFV transmission by H. marginatum ticks and evaluated how changes in temperature and biological parameters may alter the geographical range of CCHFV. In particular, variation in the rate of transovarial transmission of CCHFV in the tick produced the greatest change in CCHFV circulation in the tick population. Parameters affecting the rates of tick bite, non-systemic transmission and efficiency of tick-to-tick transmission had little effect on R0. Temperature changes that affect tick development, survival and activity rates increased the suitable area for CCHFV transmission at higher latitudes in the western Palearctic. CONCLUSIONS: Non-systemic transmission had little impact on virus transmission under all scenarios. In the area studied, increase of temperature has no impact on the routes of transmission of CCHFV. However, climate conditions favouring tick survival, which increase infected adult tick populations, together with large numbers of hosts for adults were predicted as the most likely scenario for the spread of the virus in the studied area. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed framework is able to capture the dynamics and the relative contribution of the different routes (hosts, ticks) in the transmission and spread of an important pathogen affecting human health. The high contribution of the transovarial transmission route makes the process highly dependent upon suitable hosts for adult ticks, like large domestic and wild ungulates. Climate seems to have a very reduced effect on such spread.

Tick cell lines for study of Crimean-Congo hemorrhagic fever virus and other arboviruses.

Continuous cell lines derived from many of the vectors of tick-borne arboviruses of medical and veterinary importance are now available. Their role as tools in arbovirus research to date is reviewed and their potential application in studies of tick cell responses to virus infection is explored, by comparison with recent progress in understanding mosquito immunity to arbovirus infection. A preliminary study of propagation of the human pathogen Crimean-Congo hemorrhagic fever virus (CCHFV) in tick cell lines is reported; CCHFV replicated in seven cell lines derived from the ticks Hyalomma anatolicum (a known vector), Amblyomma variegatum, Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, and Ixodes ricinus, but not in three cell lines derived from Rhipicephalus appendiculatus and Ornithodoros moubata. This indicates that tick cell lines can be used to study growth of CCHFV in arthropod cells and that there may be species-specific restriction in permissive CCHFV infection at the cellular level.

Knowledge, beliefs, and practices regarding tick bites in the Turkish population in a rural area of the Middle Anatolian Region.

The aim of this study is to determine the knowledge, beliefs, and practices of a population living in a rural area in regards to tick bites and Crimean-Congo hemorrhagic fever (CCHF). The study was conducted in a rural area located in Central Anatolia in the region of Eskisehir. A total of 1,500 individuals aged 20 years and older chosen by a stratified random sample were enrolled. A questionnaire was administered in person. In 264 (17.4%) participants, there was a history of being bitten by a tick. This rate was higher in older persons, males, married persons, and farmers. The most commonly reported protective behavior was wearing long sleeves and long pants when wandering in rural areas (65.1% of participants). The least commonly reported behavior was using insect repellent on skin or clothes (3.3% of participants). Only 799 participants (54%) had heard about CCHF as a disease associated with ticks. Females, those with primary school education, housewives, and male farmers had a high frequency of having heard about CCHF. Tick bites and CCHF are important public health problems, yet the current knowledge of these problems is not sufficient in populations living in rural areas of the Middle Anatolian Region of Turkey.

The trend towards habitat fragmentation is the key factor driving the spread of Crimean-Congo haemorrhagic fever.

We aimed to characterize an environmental niche driving the distribution of Crimean-Congo haemorrhagic fever (CCHF) in Turkey, using a geo-referenced collection of cases reported between 2003 and 2008 and a set of climate and vegetation features. We used mean monthly air temperatures and Normalized Derived Vegetation Index (NDVI) values, at a resolution of 0.1 degrees , as well as climate features at and below the surface. We computed significant differences in monthly variables between positive and negative sites, within the range of distribution of the tick vector. Seasonal climate (growth season and summer length, accumulated temperatures in winter) and vegetation components (anomalies in NDVI data) were analysed. Fragmentation of habitat was obtained from NDVI monthly data at a resolution of 1 km. Neither single climate or vegetation variables, nor any individual seasonal component, accounted in both space and time for the delineation of areas of disease although accumulated temperatures in winter consistently showed lower values in areas where the disease was reported. Coherent and significant differences between disease-containing and disease-free sites were found when habitat fragmentation and connectivity were examined. High fragmentation and connectivity were unambiguously associated with sites where disease is reported and accounted for the spatial spread of cases in 2003-2008. CCHF cases were always associated with areas of highly fragmented and well-connected patches within the range of the tick vector, while there were no reports from areas with low fragmentation. There was a linear relationship between degree of fragmentation and case incidence. The implications of these findings are discussed with reference to the concept of disease spread through networks of connected spots with high densities of infected vectors and social factors driving different human activities in sites of high fragmentation.

Immunogenicity of combination DNA vaccines for Rift Valley fever virus, tick-borne encephalitis virus, Hantaan virus, and Crimean Congo hemorrhagic fever virus.

DNA vaccines for Rift Valley fever virus (RVFV), Crimean Congo hemorrhagic fever virus (CCHFV), tick-borne encephalitis virus (TBEV), and Hantaan virus (HTNV), were tested in mice alone or in various combinations. The bunyavirus vaccines (RVFV, CCHFV, and HTNV) expressed Gn and Gc genes, and the flavivirus vaccine (TBEV) expressed the preM and E genes. All vaccines were delivered by gene gun. The TBEV DNA vaccine and the RVFV DNA vaccine elicited similar levels of antibodies and protected mice from challenge when delivered alone or in combination with other DNAs. Although in general, the HTNV and CCHFV DNA vaccines were not very immunogenic in mice, there were no major differences in performance when given alone or in combination with the other vaccines.

Type I interferon inhibits Crimean-Congo hemorrhagic fever virus in human target cells.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a causative agent of severe hemorrhagic fever occurring sporadically in parts of Africa, Asia, Southeast Europe, and the Middle East. Its recent recognition as a potential agent of bioterrorism/biowarfare highlights the need for effective antiviral therapy. In this study, it is shown that human endothelial cells are permissive to CCHFV. It is also shown that interferon-alpha inhibits the growth of CCHFV in human endothelial and hepatoma cells, reducing virus yields by a factor of 100-1,000. By using a siRNA approach, it was demonstrated that the interferon-induced MxA GTPase is a major factor mediating the antiviral effect against CCHFV, in agreement with previous findings showing that recombinant MxA inhibits CCHFV replication by interacting with the viral nucleocapsid protein. The identification of intrinsic cellular resistance factors that block CCHFV replication may help in designing novel antiviral agents.

A simple assay for determining antiviral activity against Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that is emerging as a significant human pathogen in many regions of the world, including Africa, Asia, and Europe. In this report, we describe a simple screening method for discovering new antiviral compounds directed against CCHFV. Antiviral activity was determined by assaying infected SW-13 cells (human adrenal gland carcinoma) for protection from cytopathic effect (CPE). By using an in vitro neutral red uptake assay, we were able to quantitatively measure CPE induced by CCHFV. As a proof of concept, we used this method to evaluate the antiviral activity of ribavirin and a series of structural analogs (ribamidine, 6-azauridine, selenazofurin, and tiazofurin) against four geographically diverse strains of CCHFV. Ribavirin inhibited the replication of CCHFV as reported previously using plaque reduction assays. One drug, ribamidine, showed antiviral activity that was 4.5- to 8-fold less than that of ribavirin, and the other three drugs (6-azauridine, selenazofurin, and tiazofurin) did not show significant antiviral activity. There were no significant differences in drug sensitivities among the CCHFV strains. Development of this simple and reliable assay will potentially allow high-throughput screening for discovering additional antiviral drugs to combat this important public health threat.

[Experimental transmission of Crimean-Congo hemorrhagic fever virus by Rhipicephalus evertsi evertsi (Acarina:Ixodidae)]. / Transmission expérimentale du virus de la fièvre hémorragique de Crimée-Congo par la tique Rhipicephalus evertsi evertsi (Acarina: Ixodidae).

We have conducted experiments to assess the ability of Rhipicephalus evertsi evertsi tick to transmit the Crimean-Congo haemorrhagic fever (CCHF) and determine their reproductive cycle. The Rh. e. evertsi was infected by intracoelomic (transparietal and intra-anal) inoculation during the imaginal stases and by oral feeding on an infected viremic goat during imaginal and nymphal stases. The infection rate, transovarial and trans-stasial CCHF virus transmission were monitored for virus reisolation after suckling mice inoculation and the virus identified by ELISA and IFA for antigen detection. After intracoelomic inoculation, unfed adults had viral titers ranging from 0.67 to 2.9 log DL50/0.02 ml and had transmitted the virus to their vertebrate hosts by blood feeding. After 8 to 10 days of blood feeding duration, infection rates were respectively 36% and 100% for male and female ticks. In two instances out of seven transovarial transmission was observed and the virus reisolated from larvae of first generation. However, the virus was not detected after nymphal metamorphosis. After blood feeding on viremic goats, 71% of the nymphae were infected. After metamorphoses 22% of the males and 42% of the females had a low virus titer. Rh. e. evertsi appears to have a limited efficacy in transmitting and replicating the CCHF virus but must be not neglected as a potential vector during an epizootic manifestation.

[Crimean-Congo hemorrhagic fever in Senegal. Latest data on the ecology of the CCHF virus]. / La fièvre hémorragique de Crimée-Congo au Sénégal. Dernières données sur l'écologie du virus CCHF.

The authors finalize the knowledge on the ecology of the CCHF virus in Senegal, West Africa. They specify two new major data for the understanding of the viral ecology in West Africa. The recognition of a bird species, common and widely distributed in Senegal (Tockus erythrorhynchus, Coraciiformes, Bucerotidae), that replicates the virus and infects the immature stages of its current parasite Hyalomma marginatum rufipes in more than 90% of the cases, explains why the minimum infection rate of the adults of this species of tick is always very high. The implication of Rhipicephalus evertsi evertsi in the viral ecology and/or a high efficiency of the transovarial transmission of the virus in Hy. m. rufipes would help to explain the maintenance of the endemy in the sahelian area. In the sahelian zone, Hy. marginatum rufipes must play the leading part, together with Rh. e. evertsi if vector, for the maintenance of the endemy. Hy. truncatum, the adults of which can readily bite man, ensures the vectorial transmission to him. In the sudanian zone, Amblyomma variegatum must play the same part as the Hyalomma and Rh. e. evertsi (if vector), and is surely the main vector to man, giving perhaps rise to less virulent strains (non hemorrhagic ones).

Experimental vector incompetence of a soft tick, Ornithodoros sonrai (Acari: Argasidae), for Crimean-Congo hemorrhagic fever virus.

Adults and nymphs of a soft tick, Ornithodoros sonrai Sautet & Witkowski, were allowed to feed on suckling mice that had been experimentally infected with Crimean-Congo hemorrhagic fever (CCHF) virus (IbAr 10200 strain). The mean viral titer of mouse blood at the time of tick feeding was 10(3.2) plaque-forming units (PFU) per ml. Samples of ticks were assayed on 12 occasions between days 0 and 31 after the viremic blood meal. Mean CCHF viral titers were 10(2.1) PFU per tick immediately after the viremic meal but declined to 10(1.2) PFU per tick after 2 d, and no virus was detected beyond 8 d. The percentage of ticks with detectable virus was 92% (22/24) immediately after the viremic meal, but then declined to 20% (2/10) after 4 d and to 0% (0/44) after 11 or more days. Ticks were allowed to feed on sets of three naive suckling mice on days 0, 2, 5, 8, 11, 14, 21, and 28 after the viremic blood meal, but CCHF viral transmission did not occur. Similarly, no transovarial transmission of virus from CCHF virus-exposed O. sonrai to their progeny was observed. These results strongly indicate that O. sonrai is not a vector of CCHF virus.

Transmission of Crimean-Congo haemorrhagic fever virus from experimentally infected sheep to Hyalomma truncatum ticks.

Crimean-Congo haemorrhagic fever (CCHF) virus was inoculated into West African sheep that were simultaneously infested with adult Hyalomma truncatum ticks. Certain sheep developed a viraemia and antibodies, indicating virus infection and replication; however, the length and magnitude of the viraemia and serological responses corresponded to the animals' immunological status. Tick attachment and feeding was not influenced by sheep infection. CCHF virus infection was acquired by 11-33% of female and 0-60% of male ticks. Infection in the ticks did not influence their feeding success, as judged by weight at drop-off, and the weight of eggs produced by infected and non-infected ticks was similar. Transovarial transmission of CCHF virus was demonstrated in 2 of 12 (17%) egg batches from infected female ticks, but in none of 19 egg batches from ticks that tested negative for CCHF virus. Our results suggest that under certain ecological conditions, sheep may serve to amplify CCHF virus in nature through horizontal transmission and that the maintenance cycle also may be influenced by transovarial transmission to the next generation of ticks.

Comparative studies on the vector capacity of the different stages of Amblyomma variegatum Fabricius and Hyalomma rufipes Koch for Congo virus, after intracoelomic inoculation.

Groups of nymphs and adults of Hyalomma rufipes and larvae, nymphs and adults of Amblyomma variegatum were infected by intracoelomic inoculation with Congo virus at concentrations of 10(3.5)LD50 (Group A), 10(1.5)LD50 (Group B) and 10(0.5)LD50 (Group C). The infection rates for the different groups were: Groups A, 92-100% for all stages of ticks, except for adult H. rufipes which had 87%; Group B, 56.8, 68.1 and 50.7% for larvae, nymphs and adults of A. variegatum, respectively, and 96.3 and 84.4% for nymphs and adults of H. rufipes, respectively; Group C, 0% for larvae, nymphs and adults of A. variegatum and 8 and 1.7%, respectively, for nymphs and adult adult of H. rufipes. The 1-5% infection threshold was 10(0.5)LD50 for nymphs of H. rufipes and a dose greater than this for all the other stages of the two species of ticks. The 50% infection threshold was 10(1.5)LD50 for larvae and adults of A. variegatum, but less than 10(1.5)LD50 for nymphs of A. variegatum and nymphs and adults of H. rufipes. Peak virus titres obtained for H. rufipes were 10(6.0) for Group A and B nymphs and 10(3.0) for Group A and B adults, and 10(1.0) each for Group C nymphs and adults. For A. variegatum, the peak titres for Groups A, B and C were, respectively, 10(1.0), 10(0.8), and 0 for nymphs and 10(1.5), 10(0.6) and 0 for adults. Sera from 100% (24/24) of rabbits used to feed adult H. rufipes and from 65% (31/48) used to feed nymphs and adult A. variegatum were positive for compliment fixation antibody to Congo virus at a titre of 1:8 from Days 15-35 postattachment of ticks. Viremia in rabbit was detected in 12.5% (3/25) of rabbits used to feed adult H. rufipes.