Persistence of IgG and neutralizing antibodies in Crimean-Congo hemorrhagic fever survivors.

The World Health Organization classified Crimean-Congo hemorrhagic fever (CCHF) as a high-priority infectious disease and emphasized the performance of research studies and product development against it. Little information is available about the immune response due to natural CCHF virus (CCHFV) infection in humans. Here, we investigated the persistence of IgG and neutralizing antibodies in serum samples collected from 61 Iranian CCHF survivors with various time points after recovery (<12, 12-60, and >60 months after disease). The ELISA results showed IgG seropositivity in all samples while a pseudotyped based neutralization assay findings revealed the presence of neutralizing antibody in 29 samples (46.77%). For both IgG and neutralizing antibodies, a decreasing trend of titer was observed with the increase in the time after recovery. Not only the mean titer of IgG (772.80 U/mL) was higher than mean neutralizing antibody (25.64) but also the IgG persistence was longer. In conclusion, our findings provide valuable information about the long-term persistence of humoral immune response in CCHF survivors indicating that IgG antibody can be detected at least 8 years after recovery and low titers of neutralizing antibody can be detected in CCHF survivors.

Crimean-Congo hemorrhagic fever virus prevalence in livestock of Jabalpur, Madhya Pradesh, Central India and its implications for public health.

The rise of Crimean-Congo Hemorrhagic Fever (CCHF), poses a significant global health challenge, urging immediate action and continuous surveillance. With no available vaccines, monitoring pathogen presence is critical to identify at-risk areas promptly. A study was designed to assess the incidence of CCHF virus in goats and cattle using commercial ELISA IgG kits in tribal-dominated regions. Overall, 16% of the samples (n = 63/393) were positive for CCHF virus-specific IgG antibodies, whereas sero-prevalence detected in cattle 11.6% [95% CI:7-17.7] and in goats 18.9% [95% CI: 13.76-24.01], respectively. Statistically, Animal gender and age didn't significantly affect prevalence (p-value >0.05). Our finding indicates unnoticed CCHF virus circulation. Notably, lack of public awareness about zoonotic diseases in the study region was recorded. To combat this emerging tick-borne disease effectively, it's crucial to screen individuals with hemorrhagic manifestations in healthcare settings and active surveillance of ticks to prevent unwarranted public health outbreaks and design preventive interventions.

A mRNA Vaccine for Crimean-Congo Hemorrhagic Fever Virus Expressing Non-Fusion GnGc Using NSm Linker Elicits Unexpected Immune Responses in Mice.

Crimean-Congo hemorrhagic fever (CCHF), caused by Crimean-Congo Hemorrhagic virus (CCHFV), is listed in the World Health Organization's list of priority diseases. The high fatality rate in humans, the widespread distribution of CCHFV, and the lack of approved specific vaccines are the primary concerns regarding this disease. We used microfluidic technology to optimize the mRNA vaccine delivery system and demonstrated that vaccination with nucleoside-modified CCHFV mRNA vaccines encoding GnNSmGc (vLMs), Gn (vLMn), or Gc (vLMc) induced different immune responses. We found that both T-cell and B-cell immune responses induced by vLMc were better than those induced by vLMn. Interestingly, immune responses were found to be lower for vLMs, which employed NSm to link Gn and Gc for non-fusion expression, compared to those for vLMc. In conclusion, our results indicated that NSm could be a factor that leads to decreased specific immune responses in the host and should be avoided in the development of CCHFV vaccine antigens.

Animal Exposure Model for Mapping Crimean-Congo Hemorrhagic Fever Virus Emergence Risk.

To estimate the determinants of spatial variation in Crimean-Congo hemorrhagic fever virus (CCHFV) transmission and to create a risk map as a preventive public health tool, we designed a survey of small domestic ruminants in Andalusia, Spain. To assess CCHFV exposure spatial distribution, we analyzed serum from 2,440 sheep and goats by using a double-antigen ELISA and modeled exposure probability with environmental predictors by using generalized linear mixed models. CCHFV antibodies detected in 84 samples confirmed low CCHFV prevalence in small domestic ruminants in the region. The best-fitted statistical model indicated that the most significant predictors of virus exposure risk were cattle/horse density and the normalized difference vegetation index. Model validation showed 99.7% specificity and 10.2% sensitivity for identifying CCHFV circulation areas. To map CCHFV exposure risk, we projected the model at a 1 × 1-km spatial resolution. Our study provides insight into CCHFV ecology that is useful for preventing virus transmission.

Crimean-Congo Hemorrhagic Fever Virus Diversity and Reassortment, Pakistan, 2017-2020.

Sporadic cases and outbreaks of Crimean-Congo hemorrhagic fever (CCHF) have been documented across Pakistan since 1976; however, data regarding the diversity of CCHF virus (CCHFV) in Pakistan is sparse. We whole-genome sequenced 36 CCHFV samples collected from persons infected in Pakistan during 2017-2020. Most CCHF cases were from Rawalpindi (n = 10), followed by Peshawar (n = 7) and Islamabad (n = 4). Phylogenetic analysis revealed the Asia-1 genotype was dominant, but 4 reassorted strains were identified. Strains with reassorted medium gene segments clustered with Asia-2 (n = 2) and Africa-2 (n = 1) genotypes; small segment reassortments clustered with the Asia-2 genotype (n = 2). Reassorted viruses showed close identity with isolates from India, Iran, and Tajikistan, suggesting potential crossborder movement of CCHFV. Improved and continuous human, tick, and animal surveillance is needed to define the diversity of circulating CCHFV strains in Pakistan and prevent transmission.

Crimean-Congo Hemorrhagic Fever Virus Seroprevalence in Human and Livestock Populations, Northern Tanzania.

We conducted a cross-sectional study of Crimean-Congo hemorrhagic fever virus (CCHFV) in northern Tanzania. CCHFV seroprevalence in humans and ruminant livestock was high, as were spatial heterogeneity levels. CCHFV could represent an unrecognized human health risk in this region and should be included as a differential diagnosis for febrile illness.

Approaching the complexity of Crimean-Congo hemorrhagic fever virus serology: A study in swine.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne zoonotic orthonairovirus of public health concern and widespread geographic distribution. Several animal species are known to seroconvert after infection with CCHFV without showing clinical symptoms. The commercial availability of a multi-species ELISA has led to an increase in recent serosurveillance studies as well as in the range of species reported to be exposed to CCHFV in the field, including wild boar (Sus scrofa). However, development and validation of confirmatory serological tests for swine based on different CCHFV antigens or test principles are hampered by the lack of defined control sera from infected and non-infected animals. For the detection of anti-CCHFV antibodies in swine, we established a swine-specific in-house ELISA using a panel of swine sera from CCHFV-free regions and regions with reported CCHFV circulation. We initially screened more than 700 serum samples from wild boar and domestic pigs and observed a correlation of ≃67% between the commercial and the in-house test. From these sera, we selected a panel of 60 samples that were further analyzed in a newly established indirect immunofluorescence assay (iIFA) and virus neutralization test. ELISA-non-reactive samples tested negative. Interestingly, only a subset of samples reactive in both ELISA and iIFA displayed CCHFV-neutralizing antibodies. The observed partial discrepancy between the tests may be explained by different test sensitivities, antibody cross-reactivities or suggests that the immune response to CCHFV in swine is not necessarily associated with eliciting neutralizing antibodies. Overall, this study highlights that meaningful CCHFV serology in swine, and possibly other species, should involve the performance of multiple tests and careful interpretation of the results.

Third International Conference on Crimean-Congo Hemorrhagic Fever in Thessaloniki, Greece, September 19-21, 2023.

The Third International Conference on Crimean-Congo Hemorrhagic Fever (CCHF) was held in Thessaloniki, Greece, September 19-21, 2023, bringing together a diverse group of international partners, including public health professionals, clinicians, ecologists, epidemiologists, immunologists, and virologists. The conference was attended by 118 participants representing 24 countries and the World Health Organization (WHO). Meeting sessions covered the epidemiology of CCHF in humans; Crimean-Congo hemorrhagic fever virus (CCHFV) in ticks; wild and domestic animal hosts; molecular virology; pathogenesis and animal models; immune response related to therapeutics; and CCHF prevention in humans. The concluding session focused on recent WHO recommendations regarding disease prevention, control strategies, and innovations against CCHFV outbreaks. This meeting report summarizes lectures by the invited speakers and highlights advances in the field.

Molecular identification of Crimean-Congo haemorrhagic fever virus in Hyalomma rufipes and Amblyomma variegatum in the Upper East Region of Ghana.

Sampled ticks were screened for Crimean-Congo haemorrhagic fever virus (CCHFV) using an assay that targets the nucleoprotein gene region of the S segment, a conserved region of the CCHFV genome. Minimum infection rates of 0.34% and 0.10% were obtained when testing pools of Hyalomma rufipes and Amblyomma variegatum, respectively. Next-generation sequencing and phylogenetic analysis showed that the S and L segments of the CCHFV isolate clustered with those of similar isolates of genotype III. However, analysis of the M segment showed that reassortment had occurred, causing this segment to cluster with those of isolates of genotype I, providing the first evidence of such an occurrence in Ghana.

Establishment of a lethal mouse model of emerging tick-borne orthonairovirus infections.

Emerging and reemerging tick-borne virus infections caused by orthonairoviruses (family Nairoviridae), which are genetically distinct from Crimean-Congo hemorrhagic fever virus, have been recently reported in East Asia. Here, we have established a mouse infection model using type-I/II interferon receptor-knockout mice (AG129 mice) both for a better understanding of the pathogenesis of these infections and validation of antiviral agents using Yezo virus (YEZV), a novel orthonairovirus causing febrile illnesses associated with tick bites in Japan and China. YEZV-inoculated AG129 mice developed hepatitis with body weight loss and died by 6 days post infection. Blood biochemistry tests showed elevated liver enzyme levels, similar to YEZV-infected human patients. AG129 mice treated with favipiravir survived lethal YEZV infection, demonstrating the anti-YEZV effect of this drug. The present mouse model will help us better understand the pathogenicity of the emerging tick-borne orthonairoviruses and the development of specific antiviral agents for their treatment.

Nucleocapsid protein-specific monoclonal antibodies protect mice against Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a WHO priority pathogen. Antibody-based medical countermeasures offer an important strategy to mitigate severe disease caused by CCHFV. Most efforts have focused on targeting the viral glycoproteins. However, glycoproteins are poorly conserved among viral strains. The CCHFV nucleocapsid protein (NP) is highly conserved between CCHFV strains. Here, we investigate the protective efficacy of a CCHFV monoclonal antibody targeting the NP. We find that an anti-NP monoclonal antibody (mAb-9D5) protected female mice against lethal CCHFV infection or resulted in a significant delay in mean time-to-death in mice that succumbed to disease compared to isotype control animals. Antibody protection is independent of Fc-receptor functionality and complement activity. The antibody bound NP from several CCHFV strains and exhibited robust cross-protection against the heterologous CCHFV strain Afg09-2990. Our work demonstrates that the NP is a viable target for antibody-based therapeutics, providing another direction for developing immunotherapeutics against CCHFV.

Investigating Crimean-Congo haemorrhagic fever virus seropositivity in camels and human behavioural risks in an abattoir in Nigeria.

Crimean-Congo haemorrhagic fever virus (CCHFV) is an emerging viral pathogen with pandemic potential that is often misdiagnosed. Case fatality in low-resource settings could be up to 40% due to close contact between animals and humans. A two-year cross-sectional study was conducted in Fagge abattoir, Kano State, Nigeria, to estimate the seropositivity of CCHFV in camels using a commercial multi-species competitive enzyme-linked immunosorbent assay (ELISA). A closed-ended questionnaire was administered to the abattoir workers to assess their awareness, mitigation, and behavioural practices associated with CCHF. Of the 184 camels tested, 179 (97%) were seropositive for CCHFV (95% confidence interval (CI): 93.77, 99.11). The median (interquartile range (IQR)) age of respondents was 41 (35-52), with 62% having no education. Respondents had little knowledge about CCHFV and the concept of zoonotic disease. In this study, the high estimated prevalence of antibodies to CCHFV in camels highlights the heightened risk of transmission of CCHFV in Nigeria. Similarly, a concerning lack of knowledge and inadequate preventive practices, alongside a prevalence of high-risk behaviours associated with CCHF among abattoir workers, were noted in this study. Thus, there is an urgent need for comprehensive public health education and collaborative One Health strategies to avert the threats of spillover events.

Nairovirus polymerase mutations associated with the establishment of persistent infection in human cells.

Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne virus of the Orthonairovirus genus, persistently infects tick cells. It has been reported to establish persistent infection in non-human primates, but virological analysis has not yet been performed in human cells. Here, we investigated whether and how nairoviruses persistently infect human cells using Hazara orthonairovirus (HAZV), a surrogate model for CCHFV. We established a human cell line that was persistently infected with HAZV. Surprisingly, virions of persistently infected HAZV (HAZVpi) were not observed in the culture supernatants. There were five mutations (mut1, mut2, mut3, mut4, and mut5) in L protein of HAZVpi. Mutations in L protein of HAZVpi contribute to non-detection of virion in the supernatants. Lmut4 was found to cause low viral growth rate, despite its high polymerase activity. The low growth rate was restored by Lmut2, Lmut3, and Lmut5. The polymerase activity of Lmut1 was extremely low, and recombinant HAZV carrying Lmut1 (rHAZV/Lmut1) was not released into the supernatants. However, genomes of rHAZV/Lmut1 were retained in the infected cells. All mutations (Lmut1-5) found in L protein of HAZVpi were required for experimental reproduction of HAZVpi, and only Lmut1 and Lmut4 were insufficient. We demonstrated that point mutations in viral polymerase contribute to the establishment of persistent HAZV infection. Furthermore, innate immunity was found to be suppressed in HAZVpi-infected cells, which also potentially contributes to viral persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells. IMPORTANCE: We investigated whether and how nairoviruses persistently infect human cells, using Hazara orthonairovirus (HAZV), a surrogate model for Crimean-Congo hemorrhagic fever virus. We established a human cell line that was persistently infected with HAZV. Five mutations were found in L protein of persistently infected HAZV (HAZVpi): mut1, mut2, mut3, mut4, and mut5. Among them, Lmut1 and Lmut4 restricted viral growth by low polymerase activity and low growth rate, respectively, leading to inhibition of viral overgrowth. The restriction of viral growth caused by Lmut1 and Lmut4 was compensated by other mutations, including Lmut2, Lmut3, and Lmut5. Each of the mutations found in L protein of HAZVpi was concluded to cooperatively modulate viral growth, which facilitates the establishment of persistent infection. Suppression of innate immunity also potentially contributes to virus persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells.

Neutralizing monoclonal antibodies against the Gc fusion loop region of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne virus, prevalent in more than 30 countries worldwide. Human infection by this virus leads to severe illness, with an average case fatality of 40%. There is currently no approved vaccine or drug to treat the disease. Neutralizing antibodies are a promising approach to treat virus infectious diseases. This study generated 37 mouse-derived specific monoclonal antibodies against CCHFV Gc subunit. Neutralization assays using pseudotyped virus and authentic CCHFV identified Gc8, Gc13, and Gc35 as neutralizing antibodies. Among them, Gc13 had the highest neutralizing activity and binding affinity with CCHFV Gc. Consistently, Gc13, but not Gc8 or Gc35, showed in vivo protective efficacy (62.5% survival rate) against CCHFV infection in a lethal mouse infection model. Further characterization studies suggested that Gc8 and Gc13 may recognize a similar, linear epitope in domain II of CCHFV Gc, while Gc35 may recognize a different epitope in Gc. Cryo-electron microscopy of Gc-Fab complexes indicated that both Gc8 and Gc13 bind to the conserved fusion loop region and Gc13 had stronger interactions with sGc-trimers. This was supported by the ability of Gc13 to block CCHFV GP-mediated membrane fusion. Overall, this study provides new therapeutic strategies to treat CCHF and new insights into the interaction between antibodies with CCHFV Gc proteins.

Detection of Crimean-Congo haemorrhagic fever virus in Hyalomma marginatum ticks, southern France, May 2022 and April 2023.

Crimean-Congo haemorrhagic fever (CCHF), a potentially severe zoonotic viral disease causing fever and haemorrhagic manifestations in humans. As the Crimean-Congo haemorrhagic fever virus (CCHFV) has been detected in ticks in Spain and antibodies against the virus in ruminant sera in Corsica, it was necessary to know more about the situation in France. In 2022-2023, CCHFV was detected in 155 ticks collected from horses and cattle in southern France.

Secondary multilevel mixed-effects modelling of seroprevalence trends of Crimean-Congo haemorrhagic fever.

Background: Some review papers and meta-analyses have investigated seroprevalence and fatality trends of the Crimean-Congo hemorrhagic fever (CCHF), but it is not clear if its seroprevalence is increasing. Aim: To investigate the trend in the seroprevalence of CCHF. Methods: We conducted a secondary analysis of the results of a meta-analysis of the seroprevalence of CCHF published in 2019. We used a multilevel mixed effects Poisson regression to find the predictors of seropositivity. To explain the magnitude effect, we reported an incidence rate ratio (IRR) with a 95% confidence interval (CI). We conducted multilevel modeling using Stata 14 for data analysis. Results: In the fixed effects model, time was significantly associated with increased seropositivity (IRR = 1.025, 95% CI = 1.021-1.030), and no significant association was found for local sampling (IRR = 1.026, 95% CI = 0.988-1.065). In the mixed effects model, random intercepts of the country and parallel of latitude were applied as 3 levels of the model (prevalence rate of each study, nested within countries and latitude parallel). Accordingly, time was significantly associated with a reduction of seropositivity (IRR = 0.899, 95% CI = 0.891-0.907), and local sampling was significantly associated with increased seropositivity (IRR = 2.477, 95% CI = 2.316-2.649). Conclusion: Despite reporting increasing trends for seroprevalence of CCHF in previous reviews and the fixed effects model of the present study, the secondary mixed effects modeling showed a decreasing trend. The multilevel generalized model is recommended for such temporal and spatial designs in the future.

Emergence of Crimean-Congo Hemorrhagic Fever Virus in Eastern Senegal in 2022.

Crimean-Congo hemorrhagic fever (CCHF), the most widespread tick-borne viral human infection, poses a threat to global health. In this study, clinical samples collected through national surveillance systems were screened for acute CCHF virus (CCHFV) infection using RT-PCR and for exposure using ELISA. For any CCHF-positive sample, livestock and tick samples were also collected in the neighborhood of the confirmed case and tested using ELISA and RT-PCR, respectively. Genome sequencing and phylogenetic analyses were also performed on samples with positive RT-PCR results. In Eastern Senegal, two human cases and one Hyalomma tick positive for CCHF were identified and a seroprevalence in livestock ranging from 9.33% to 45.26% was detected. Phylogenetic analyses revealed that the human strain belonged to genotype I based on the available L segment. However, the tick strain showed a reassortant profile, with the L and M segments belonging to genotype I and the S segment belonging to genotype III. Our data also showed that our strains clustered with strains isolated in different countries, including Mauritania. Therefore, our findings confirmed the high genetic variability inside the CCHF genotypes and their introduction to Senegal from other countries. They also indicate an increasing CCHF threat in Senegal and emphasize the need to reinforce surveillance using a one-health approach.

Molting incidents of Hyalomma spp. carrying human pathogens in Germany under different weather conditions.

BACKGROUND: Hyalomma marginatum and H. rufipes are two-host tick species, which are mainly distributed in southern Europe, Africa to central Asia but may also be found in Central and Northern Europe through introduction by migratory birds. METHODS: Ticks were collected while feeding or crawling on animals and humans, or from the environment, in different regions in Germany, between 2019 and 2021 in a citizen science study and from 2022 to 2023 in the wake of this study. RESULTS: From 2019 to 2023, a total of 212 Hyalomma adult ticks were detected in Germany. This included 132 H. marginatum and 43 H. rufipes ticks sent to research institutions and 37 photographic records that were only identified to genus level. The number of detected ticks varied over the years, with the highest number of 119 specimens recorded in 2019, followed by 57 in 2020. Most of the specimens were collected from horses, while some were collected from other animals, humans or found crawling on human clothes or other objects inside or outside houses. The screening of 175 specimens for Crimean-Congo hemorrhagic fever virus and of 132 specimens for Babesia/Theileria spp. by PCR gave negative results, while human-pathogenic Rickettsia were detected in 44% (77/175) of the total samples. Subsequent amplicon sequencing and phylogenetic analysis of representative samples determined the species of 41 Rickettsia aeschlimannii and one R. slovaca sequences. CONCLUSIONS: Analysis of climatic factors indicated a significantly higher probability of Hyalomma occurrence at locations with higher average spring temperature during the years 2019 and 2020 compared to randomly generated pseudo-absence locations. Dry and hot conditions probably facilitated Hyalomma nymphs' survival and molting into adults during these years.

LDLR is an entry receptor for Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread tick-born zoonotic bunyavirus that causes severe hemorrhagic fever and death in humans. CCHFV enters the cell via clathrin-mediated endocytosis which is dependent on its surface glycoproteins. However, the cellular receptors that are required for CCHFV entry are unknown. Here we show that the low density lipoprotein receptor (LDLR) is an entry receptor for CCHFV. Genetic knockout of LDLR impairs viral infection in various CCHFV-susceptible human, monkey and mouse cells, which is restored upon reconstitution with ectopically-expressed LDLR. Mutagenesis studies indicate that the ligand binding domain (LBD) of LDLR is necessary for CCHFV infection. LDLR binds directly to CCHFV glycoprotein Gc with high affinity, which supports virus attachment and internalization into host cells. Consistently, a soluble sLDLR-Fc fusion protein or anti-LDLR blocking antibodies impair CCHFV infection into various susceptible cells. Furthermore, genetic knockout of LDLR or administration of an LDLR blocking antibody significantly reduces viral loads, pathological effects and death following CCHFV infection in mice. Our findings suggest that LDLR is an entry receptor for CCHFV and pharmacological targeting of LDLR may provide a strategy to prevent and treat Crimean-Congo hemorrhagic fever.