Crimean-Congo Hemorrhagic Fever: A Neglected Priority

Crimean-Congo Hemorrhagic Fever (CCHF) is a viral disease, which has a mortality rate of ~%3-%30. The disease is caused by a Nairovirus named CCHF virus (CCHFV), which is transmitted to humans by tick bites or by contact with animal blood. CCHF cases were seen in Southeastern Europe, Middle East, Africa, and Asia¹. In Turkey, there have been over 10.000 CCHF cases with an average fatality rate of %5, and this tick-borne disease remains to be a critical public health problem for Turkey. Especially, the farmers suffer from this disease, as CCHF and ticks are mostly widespread in rural areas². Characterizing the tick and CCHF virus strains the ticks carry is crucial to understand how the virus transmits to humans and animals. Thus, understanding the potential risks in the areas where ticks are present. 

Characterizing the genomes of ticks can help understanding the genomic links between species, reveal the genetic reasoning of their habitat of choice, and can give insights on virus capacity of these ticks. Moreover, transmission of CCHFV to humans from ticks can be understood better by characterizing the tick and CCHFV genomes.

To characterize the CCHFV and Tick genomes in Turkey, a several step action plan is needed.

Overview of the action plan steps:

• Determining the regions in Turkey where ticks, CCHF cases and tick bites are prevalent

• Collecting ticks from the determined areas

• Collecting human sera samples from the CCHF cases in Turkey (together with ticks if possible)

• Extracting genetic material from ticks and CCHFV

• Sequencing the whole genome of ticks and CCHFV

• Comparative genomics analysis 

• Region – Tick – Virus mapping

• Further bioinformatics analysis

• Planning and applying preventative measures based on the outcomes

• Continuous tracking of CCHF cases in the risky and potentially risky regions

Rural areas that farmers do animal husbandry, and perform agricultural activities, areas with high temperatures for at least a part of the year, and areas where CCHF cases were mostly seen such as Tokat, Sivas, Gümüşhane, Erzincan, Erzurum, and Çorum in Turkey could be initial candidates for sample collection². The aforementioned regions have most of the traits above, as these traits drive tick questing. Another possibility which is common is ticks transmitting from the livestock animals. Changes in land properties is also a main factor that affects CCHF case rate, and it is more influential than animals or climate².

Ticks will be collected from vegetation and livestock, domestic and wild animals. Main tick species that cause CCHF in Turkey are ixodid ticks (hard ticks). Hyalomma genus ticks are the main vectors for CCHFV, however, it is speculated that Rhipicepalus and Dermacentor may have an effect on CCHF transmission indirectly or directly, the complete role these species play in CCHF transmission is not known³. Collecting sera from CCHF cases with humans (with consent) is key too, as it shows which virus, and tick strains are connected and can transmit the virus to humans, and how fatal/dangerous that particular virus strains are.

 The key part of this action plan is to produce near-complete and accurate sequence data of ixodid tick strains and CCHFV viruses collected from the selected regions in a standardized way. This can be achieved by combining the de novo sequencing and resequencing, Next-Generation Sequencing (NGS) technologies for tick genomes. Small whole genome sequencing technology can be used to determine the CCHF virus genomes. 

Key to Tackle CCHF: Sequence & Analyze

As sequencing technologies advance, sequencing of whole genomes of organisms became faster and cheaper. These improvements led genomics tools to be used in tackling many diseases by determining the whole genomic information of microbes, the hosts that carry them and the differences caused by mutations between the same organisms⁴. Recently, fast and accurate sequencing of the SARS-CoV-2 strain sequences led researchers to design mRNA vaccines in a short time⁵ and helped keeping track of different strains⁶. Hence, the differences between these strains helped to predict the outbreaks and according to these predictions, preventative measures were taken to reduce the potential cases, and the death toll. CCHF cases mostly appear in endemic outbreaks, and it can be classified as a neglected and a tropical disease. Thus, genomic data on CCHF virus and the vector ticks is limited. Currently, there are a total of 2036 strain entries and 691 complete segments in the Virus Pathogen Database (VPD) (for Large (L) segment of CCHFv genome). For CCHF, characterizing the whole genome of the ticks that act as vectors to the CCHF virus, and the different CCHF virus strains with the latest sequencing technology will pave the way to reduce the cases and deaths caused by CCHF. Hyalomma marginatum ticks are the main vectors for CCHFV in Turkey, and these ticks do not have their whole genome sequenced. In contrast, Hyalomma asiaticum, a tick species which is a vector for Tamdy Virus (TAMV), its whole genome de novo sequenced⁷. For better understanding of CCHF transmission, de novo sequencing the whole genome of Hyalomma marginatum will be a solid reference for resequencing the ticks collected based on the de novo reference sequence. 

De novo sequencing is a technique where the genome sequencing is done without a reference genome. This can be due to lack of reference genomes, or the need of a high-quality reference genome to use for further resequencing of different genomes. De novo sequencing is also advantageous for finishing the missing gaps in a genome, identifying variations in sequences and fully characterizing the repeating regions in the sequence to do the de novo assembly in a more accurate manner⁸. In the CCHF case, hyalomma marginatum is the main vector, thus it needs to be characterized in an accurate way from scratch to get significant results while analyzing the vector-virus relationship. Moreover, an accurate reference sequence will help getting better results in resequencing of other collected ticks’ genome. In addition to hyalomma marginatum, Rhipicephalus bursa, Rhipicephalus sanguineus, and Dermacentor marginatus can be de novo sequenced as these species may have effects on rates of CCHF cases even if they are not the main species that cause CCHF in Turkey. Rhipicephalus sanguineus’ whole genome was sequenced by Jia et al. with de novo sequencing⁷, but de novo sequencing the Rhipicephalus sanguineus in Turkey may show the genomic differences between the sample tick Jia et al. used and the sample collected in Turkey. 

To perform de novo sequencing, larvaes of the determined species are needed, and the DNA of these larvaes  should be extracted and used in sequencing. Using these DNA, short DNA fragment libraries can be produced. Approximately 110 Gb of sequencing data can be generated from each tick species⁷. Genome assembly with different kinds of fragments (e.g. long insert, paired end) can be used as a complementary approach to increase accuracy. After the assembly,  further trimming must be done with various bioinformatics softwares. Based on these de novo sequences, other collected ticks can be resequenced. Ticks collected from the sera with the virus, or the ticks that already contain pathogen or pathogen remainders can be checked to analyze what makes them the vector of for their pathogens. These pathogens can be isolated and sequenced to determine and compare their sequences with other CCHFv pathogens to analyze the differences that cause them to be in that particular vector. Linkage between the virus-ticks can give crucial insights about the vector capacity-genome relations. In addition to the genomic technologies, characterization of the ticks can be done morphologically initially. However, morphological characterization is not as trustable as genomic characterization as it can be misleading in some cases, and this is the case for some classification done, and there have been research done that fix these wrong classifications with sequencing and comparative genomics⁹. After the sequencing and the characterizations of the ticks and CCHFv strains are done, CCHFv strains and ticks’ can be mapped to the regions they were initially collected. Based on the genomic data and the regional mapping, predictions can be made on the future outbreak risks. As the characterization of the ticks are made, and the relevant data of what kind of ticks are vectors to which strains of viruses, it is possible to create a risk profile of the regions. Moreover, the data collected from the human sera will provide the relevant information on the virus strains that can transmit to humans. These strains can be compared to the extracted CCHFv viruses from questing ticks, and this can give key information about which strains of CCHF viruses are in that region which have a possibility of transmitting to humans. Also, there are CCHF viruses that can transmit to some animals (particularly livestock animals) but cannot transmit to humans. Even a single mutation in the CCHF virus genome can have drastic effects on the infectivity of the virus against humans¹⁰. Thus, comparing these genomes would give a better outlook of how the CCHFv evolved to infect humans, and the risk analysis can be conducted with this information in terms of mutation possibilities that will let impaired CCHFv to infect humans. Continuous collecting and tracking of ticks in the regions and tracking of CCHF virus cases with sequence data is also important to track the changes, refine the predictions, and analyze the data to make risk analysis on these areas. Another aspect of CCHF that makes continuous tracking key is that the disease epidemiology is highly influenced by the animal migration, animal population changes, and climate changes. CCHF cases peak in summer where the weather is hot, and agricultural activities are at its maximum². Hence, sample collections and predictions for the summer period is especially important to reduce the case and death-toll related to CCHF. The predictions on tick distribution, virus strain types present in the area, potential new variants of the virus, migratory birds that carry ticks’ presence in the region, livestock animal population, overall climate in the area, vegetation changes in the area, and potential virus strains that can be dangerous in terms of mortality and infectivity in the area, and tick diversity in the area data combined together can give essential information to create predictions on the overall riskiness, and risk changes in the regions. These risk analyses can help taking preventative measures against CCHF. For instance, if an outbreak is predicted, the farmers in that region can be informed about the potential risks, trained on how to work in areas where there is tick presence, and tick host livestock animal husbandry can be limited for some terms to deal with tick risks. As data accumulates, predictions can be expanded to the areas where there are no CCHF cases seen to calculate whether is it possible to see CCHF, more tick quests and animal migration in the future that will lead to an outbreak. The genomic data and correct classification of the ticks is critical in this case too, as one of the key parts of these analyses include the profiles of the ticks that will them to be present in the currently tick-free region. 

Currently, most genomic efforts were done on CCHF virus genomes, and a significant portion of these genomes are incomplete due to limited budgets. There is also limited research done on tick genomes that cause CCHF, which is as crucial as the viruses themselves, as they are the vectors for these viruses, and they are key for transmission to humans with bites. Thus, the genomic research done using de novo genome sequencing technology together with resequencing would drastically benefit tackling the CCHF problem efforts in Turkey. 

References

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3. Gargili, A. et al. The role of ticks in the maintenance and transmission of Crimean-Congo hemorrhagic fever virus: A review of published field and laboratory studies. Antiviral Res. 144, 93–119 (2017).

4. Radford, A. D. et al. Application of next-generation sequencing technologies in virology. J. Gen. Virol. 93, 1853 (2012).

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7. Jia, N. et al. Large-Scale Comparative Analyses of Tick Genomes Elucidate Their Genetic Diversity and Vector Capacities. Cell 182, 1328-1340.e13 (2020).

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9. Hekimoğlu, O., Sağlam, İ. K., Özer, N. & Estrada-Peña, A. New molecular data shed light on the global phylogeny and species limits of the Rhipicephalus sanguineus complex. Ticks Tick. Borne. Dis. 7, 798–807 (2016).

10. Hua, B. L. et al. A single mutation in Crimean-Congo hemorrhagic fever virus discovered in ticks impairs infectivity in human cells. Elife 9, 1–18 (2020).