Novel Vaccinology Approaches against Ticks

Background and Aim: The CCHF (Crimean-Congo hemorrhagic fever) virus causes a severe disease in human with a case fatality rate of up to 50%. Since, there is no specific treatment or approved vaccine against CCHF viral infections, an accurate and early detection as well as a reliable surveillance and quantitative determination of viral load is necessary for patient improvement and case management. In this research, our aim was to develop a probe based one-step real-time reverse-transcription polymerase chain reaction (rRT-PCR) assay for in-house quantitative detection of CCHF virus. Methods: At first, the highly conserved S-fragment sequence of CCHF virus genome was adapted from GenBank and the specific probe and primers targeting this region were designed. Then, viral RNAs were extracted from 37 blood samples of different patients from east of Iran (Zahedan). The specificity and sensitivity of the probe and primers were also evaluated in positive blood samples, confirmed to have CCHF virus. A standard (PTG19-T vector containing S-fragment) for quantization was also constructed and the viral load was determined in some of positive samples. Results: From a total of 37 suspicious blood samples, 15 samples were confirmed to be positive for CCHF virus by this probe based one-step rRT-PCR assay and no false-positive result was detected according to sequencing data. The predicted fragment of 176 bp was also confirmed in all positive samples by gel-based electrophoresis analysis. The assay was linear between 10 to 103 copy numbers per each microliter of extracted plasmid for this technique and the viral load determined in one of patient blood samples was 55,000 viral particles per each milliliter, for example. Bioinformatics and experimental evaluations approved the specificity of this assay. The LOD of the assay was 10 (or fewer) copy numbers of viral genome per each microliter of the extracted genome. Conclusions: This research showed that the developed probe based one-step rRT-PCR assay is a specific, rapid, sensitive and the simple tool for detection and viral load determination of the CCHF virus. Keywords: CCHF Virus, Real-time RT-PCR, Quantitative, viral load, S-region.

Several viral hemorrhagic fever (VHF) diseases affect large populations in India. Among these, Dengue is the most common endemic mosquito-borne VHF disease. Two other common VHF diseases are tick-borne: Kyasanur Forest disease (KFD) and Crimean Congo hemorrhagic fever (CCHF). CCHF was detected in India recently. KFD virus has a wide range of tick vector species and involves several mammalian hosts. Until 2011, KFD was seen only in southern India, but recently its presence was confirmed in the Western Ghats region, including Karnataka, Tamil Nadu, Kerala, Goa and Maharashtra states. This discovery has raised suspicion whether it existed earlier in these areas. Although, the risk of human-to-human transmission of this disease is very low, it is important to consider the high prevalence of the vectors namely ticks; small mammals, monkeys, and domestic animals that are carriers of infected ticks as well as deforestation. Knowledge of KFD pathogenicity, improved and cost-effective diagnostics, laboratory networks, increased awareness of travelers to forested areas, and surveillance are needed to reduce fatality. The use of standard biosafety practices in handling the virus in the laboratory and in associated field work reduces the risk associated with infection.

lacked adjustment for confounding, we think that the results of Bacci et al.

Emergence and re-emergence of several pathogens have been witnessed by this century in the form of outbreaks, epidemics and pandemics. In India, the influencing factor that promotes dissemination of emerging and re-emerging viral infections is the biogeographical zones: a megadiverse country, characterized by varied geographical, climatic conditions and ever-changing socio-economical and geopolitical issues. These influence the movement of humans and animals and add layers of complexity for the identification and timely management of infectious diseases. This review focuses on two tick-borne infections: Crimean-Congo haemorrhagic fever (CCHF) and Kyasanur forest disease (KFD). In the last two decades, these viruses have emerged and caused outbreaks in different parts of India. KFD virus was initially identified in 1957 and was known to be endemic in Karnataka State while CCHF virus was first identified during 2010 in Gujarat State, India. These viruses have managed to emerge in new areas within the last decade. With changing epidemiology of these arboviruses, there is a probability of the emergence of these viruses from new areas in future. The investigations on these two diseases under the One Health focus involved early detection, quickly developing diagnostic tools, identifying stakeholders, capacity building by developing collaboration with major stakeholders to understand the epidemiology and geographical spread in domestic animal reservoirs and tick vectors in the affected areas, developing laboratory network, providing diagnostic reagents and biosafety and laboratory diagnosis training to the network laboratories to control these diseases.

A preliminary ecological profile of Kyasanur Forest disease virus hosts among the mammalian wildlife of the Western Ghats, India

Transmission of crimean-congo haemorrhagic fever virus from experimentally infected sheep to hyalomma truncatum ticks

Introduction: Systematic studies of endemic zoonotic diseases on the Crimean Peninsula resumed in 2015 after a long break. Objective: To specify boundaries and determine epizootiological activity of the natural foci of infectious diseases in the Crimea and to update the list of possible reservoir hosts and vectors based on the results of field studies conducted in 2015–2022. Materials and methods: Counting and trapping of small mammals, ixodid ticks and mosquitoes, collecting of pellets of birds of prey, sampling of water and other zoological and entomological materials were carried out in the Crimea in the years 2015 to 2022. Results and discussion: According to the results of epidemiological and epizootiological monitoring of the Crimean Peninsula, the samples tested positive for tularemia were collected on the territory of 14 administrative entities, leptospirosis – 21, hantaviruses – 12, tick-borne encephalitis – 2, Crimean-Congo hemorrhagic fever – 7, Lyme borreliosis – 21, West Nile fever – 3, Q fever – 2, Marseilles fever – 6, monocytic ehrlichiosis – 1, and granulocytic anaplasmosis – 8 entities. A new hantavirus was identified in the Leninsky district. Positive findings of markers of the Crimean-Congo hemorrhagic fever virus were registered not only in the main vector, but also in other species of ixodid ticks. True boundaries of the natural foci of Lyme borreliosis in the Crimea were much wider and ran through both forest and steppe zones. A stable reproducing population of epidemiologically significant Ae. albopictus mosquitoes was found on the southern coast of Crimea. Conclusions: Based on the findings, we specified the boundaries and confirmed the activity of natural foci of infectious diseases during the study period, updated sources of infections, reservoir hosts and vectors, and compiled the “List of enzootic (endemic) territories of the Crimea, on which circulation of infectious agents common to humans and animals was found based on the results of field material studies in 2015–2022,” all facilitating managerial decisions on prevention of natural focal infections.

ABSTRACTAlkhumra hemorrhagic fever virus (AHFV), a relatively new member of the Flaviviruses, was discovered in Saudi Arabia 23 years ago. AHFV is classified in the tick-borne encephalitis virus serocomplex, along with the Kyasanur forest disease virus (KFDV) and tick-borne encephalitis virus (TBEV). Currently, very little is known about the pathologies of AHFV. In this study, using the available genome information of AHFV, KFDV and TBEV, we have predicted and compared the following aspects of these viruses: evolution, nucleotide and protein compositions, recombination, codon frequency, substitution rate, N- and O-glycosylation sites, signal peptide and cleavage site, transmembrane region, secondary structure of 5’ and 3’ UTRs and RNA-RNA interactions. Additionally, we have modeled the 3D protease and RNA-dependent RNA polymerase structures for AHFV, KFDV and TBEV. Recombination analysis showed no evidence of recombination in the AHFV genome with that of either KFDV or TBEV, although single break point analysis showed that nucleotide position 7399 (in the NS4B) is a breakpoint location. AHFV, KFDV and TBEV are very similar in terms of codon frequency, the number of transmembrane regions, properties of the polyprotein, RNA-RNA interaction sequences, NS3 protease and NS5 polymerase structures and 5’ UTR structure. Using genome sequences, we showed the similarities between these closely- related viruses on several different areas.

The present manuscript deals with experimental infections of bonnet macaques (Macaca radiata) to study disease progression for better insights into the Kyasanur Forest Disease (KFD) pathogenesis and transmission. Experimentally, 10 monkeys were inoculated with KFD virus (KFDV) (high or low dose) and were regularly monitored and sampled for various body fluids and tissues at preset time points. We found that only 2 out of the 10 animals showed marked clinical signs becoming moribund, both in the low dose group, even though viremia, virus shedding in the secretions and excretions were evident in all inoculated monkeys. Anti-KFDV immunoglobulin (Ig)M antibody response was observed around a week after inoculation and anti-KFDV IgG antibody response after two weeks. Anaemia, leucopenia, thrombocytopenia, monocytosis, increase in average clotting time, and reduction in the serum protein levels were evident. The virus could be re-isolated from the skin during the viremic period. The persistence of viral RNA in the gastrointestinal tract and lymph nodes was seen up to 53 and 81 days respectively. Neuro-invasion was observed only in moribund macaques. Re-challenge with the virus after 21 days of initial inoculation in a monkey did not result in virus shedding or immune response boosting.

Objective of the study is to analyze epidemiological and epizootiological manifestations of natural-focal infections in the territory of the North-Caucasian Federal District (NCFD) in 2015. Materials and methods. Utilized were the reports of the Rospotrebnadzor Administrations, “Centers of Hygiene and Epidemiology” in the NCFD entities, Stavropol Research Anti-Plague Institute, Dagestan and Kabardino-Balkaria Plague Control Stations. Data processing was conducted using Excel Microsoft ware. Results and conclusions. Identified have been 8 nosological forms of natural-focal infections, including infections of bacterial etiology – 6 (75 %), and viral etiology – 2 (25 %). The majority of patients (164) are identified in the Stavropol Region: 67 (intestinal yersiniosis), 43 (Crimean–Congo hemorrhagic fever), 40 (Lyme borreliosis), 9 (leptospirosis), 2 (pseudotuberculosis), 1 (rabies), 1 (human granulocytic anaplasmosis) and 1 (human monocytic ehrlichiosis). The patients with 2 nosological forms of natural focal infections are identified in the Republic of Dagestan: 2 – Crimean–Congo hemorrhagic fever and one patient – tularemia. Also 2 nosological forms of natural focal infections are revealed in the Republic of Karachay-Cherkessia: one case of leptospirosis and one case of Crimean–Congo hemorrhagic fever (imported from the Stavropol region). 8502 field samples have been tested for the presence of markers of 12 nosological forms of natural-focal infections. Positive results for 10 of them have been obtained. The most comprehensive epizootiological monitoring has been carried out in the Stavropol Territory (10 nosological forms), whereat circulation of 8 pathogenic agents is registered.

Crimean Congo hemorrhagic fever (CCHF) orthonairovirus and Q fever are zoonotic infections. CCHF is the dominant tick borne viral infection among livestock, with a serious threat to humans as well. Wide host range and asymptomatic traits has made infection eradication difficult. In addition, low infectious dose of the virus has made it a highly pathogenic agent. CCHF is recognized as a threat to public health and public health services for its epidemic potential, high fatality rate (up to 40%), its potential for nosocomial outbreaks and difficulties in treatment, prevention and control. Q fever is also a serious health threating disease. Knowledge of causative agent ( Coxiella burnetii ) remains limited until today. The rate of CCHF and Q fever as zoonotic infections has remained high in most of regions worldwide. Hence implementation of accurate control strategies can help prevent its spread. Use of ticks’ repellants, control of livestock and wildlife movements and multinational policies are also essential in this regard. Noticeably, it is necessary to establish and empower the international surveillance fulfills to better predict, investigate and control these infectious diseases, improve the international public health infrastructure, develop enhanced international guidelines, and recommendations to improve the international capabilities toward restriction of disease outbreaks with adequate medical and scientific resources and expertise. To predict and monitor the CCHF and Q fever, regional rather than countrywide scale cooperation is necessary. Immigration/travel also remains an important factor influencing the spread of these agents. Key words: Crimean Congo hemorrhagic fever, Q fever, Zoonoses, control strategies

Convalescent human plasma candidate reference materials protect against Crimean-Congo haemorrhagic fever virus (CCHFV) challenge in an A129 mouse model

Crimean-Congo hemorrhagic fever (CCHF) is a medically relevant tick-borne viral disease caused by the Bunyavirus, Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is endemic to Asia, the Middle East, South-eastern Europe, and Africa and is transmitted in enzootic cycles among ticks, mammals, and birds. Human infections are mostly subclinical or limited to mild febrile illness. Severe disease may develop, resulting in multi-organ failure, hemorrhagic manifestations, and case-fatality rates up to 30%. Despite the widespread distribution and life-threatening potential, no treatments have been approved for CCHF. Antiviral inhibitory peptides, which antagonize viral entry, are licensed for clinical use in certain viral infections and have been experimentally designed against human pathogenic bunyaviruses, with in vitro and in vivo efficacies. We designed inhibitory peptides against CCHFV with and without conjugation to various polyethylene glycol and sterol groups. These additions have been shown to enhance both cellular uptake and antiviral activity. Peptides were evaluated against pseudotyped and wild-type CCHFV via neutralization tests, Nairovirus fusion assays, and cytotoxicity profiling. Four peptides neutralized CCHFV with two of these peptides shown to inhibit viral fusion. This work represents the development of experimental countermeasures for CCHF, describes a nairovirus immunofluorescence fusion assay, and illustrates the utility of pseudotyped CCHFV for the screening of entry antagonists at low containment settings for CCHF.

Regulation of apoptosis during infection has been observed for several viral pathogens. Programmed cell death and regulation of apoptosis in response to a viral infection are important factors for host or virus survival. It is not known whether Crimean-Congo hemorrhagic fever virus (CCHFV) infection regulates the apoptosis process in vitro. This study for the first time suggests that CCHFV induces apoptosis, which may be dependent on caspase-3 activation. This study also shows that the coding sequence of the S segment of CCHFV contains a proteolytic cleavage site, DEVD, which is conserved in all CCHFV strains. By using different recombinant expression systems and site-directed mutagenesis, we demonstrated that this motif is subject to caspase cleavage. We also demonstrate that CCHFV nucleocapsid protein (NP) is cleaved into a 30-kDa fragment at the same time as caspase activity is induced during infection. Using caspase inhibitors and cells lacking caspase-3, we clearly demonstrate that the cleavage of NP is caspase-3-dependent. We also show that the inhibition of apoptosis induced progeny viral titers of ∼80-90%. Thus, caspase-3-dependent cleavage of NP may represent a host defense mechanism against lytic CCHFV infection. Taken together, these data suggest that the most abundant protein of CCHFV, which has several essential functions such as protection of viral RNA and participation in various processes in the replication cycle, can be subjected to cleavage by host cell caspases.

Crimean-Congo hemorrhagic fever (CCHF) is a potentially fatal systemic disease in children caused by a tick- borne virus. Many different clinical and laboratory findings are seen in CCHF. We report here an atypical presentation of CCHF with hyponatremia. CCHF with electrolyte imbalance is not reported before. A 4-year-old girl presented with fever, fatigue and unconsciousness with hyponatremia. Based on the clinical and epidemiological findings, virus infection was suspected. Hyponatremia is has never been reported in Crimean-Congo hemorrhagic fever (CCHF), as was observed in this case. The diagnosis was confirmed by detection of IgM antibody to CCHF virus and positive Real-Time PCR. We report the first case of imported CCHF presenting as hyponatremia. This electrolyte imbalance has never been reported before in CCHF in children, and the clinician should consider this entity in complications to explain unconsciousness.