Crimean-Congo hemorrhagic fever virus in ticks collected from livestock in Albania

Albania is a Balkan country endemic for Crimean-Congo hemorrhagic fever (CCHF). It was shown previously that CCHF virus (CCHFV) sequences from Albanian patients cluster into Europe 1 clade. Aim of the present study was to test for CCHFV ticks collected in several regions of Albania, and to determine the genetic lineage(s) of the CCHFV strains in relation with their geographic distribution. A total of 726 ticks (366 Hyalomma marginatum, 349 Rhipicephalus bursa and 11 Rhipicephalus sanguineus) collected from livestock during 2007–2014 were included in the study. Thirty of 215 (13.9%) tick pools were positive for CCHFV. Lineage Europe 1 was detected in H. marginatum ticks collected in the endemic region of Albania, while lineage Europe 2 was detected mainly in R. bursa ticks in various regions of the country. Both genetic lineages were detected in the CCHF endemic area (northeastern Albania), while only Europe 2 lineage was detected in the south of the country. A higher genetic diversity was seen among Europe 2 than Europe 1 Albanian sequences (mean distance 3.7% versus 1%), suggesting a longer evolution of AP92-like strains (Europe 2) in their tick hosts. The present study shows that besides CCHFV lineage Europe 1, lineage Europe 2 is also present in Albania. Combined with results from recent studies, it is concluded that lineage Europe 2 is widely spread in the Balkans and Turkey, and is associated mainly with R. bursa ticks (at least in this region). Its pathogenicity and impact to the public health remain to be elucidated.     

Detection of Crimean-Congo hemorrhagic fever virus in ticks collected from South Russia

Crimean-Congo hemorrhagic fever (CCHF) has a central role among tick-borne infections in southern Russia. Multiple cases of disease are recorded annually in most regions with the СCHF foci; moreover, an expansion of the geographic range of the disease has been noted. Since 1999, more than 2300 people have fallen ill in Russia. Ticks are the main vectors and reservoirs of the Crimean-Congo hemorrhagic fever virus (CCHFV). As it is currently known, CCHFV has been detected in ticks of 33 species. In the period 2012–2019 in the south of the European part of Russia, more than 38,000 ticks of 14 species of the genera Hyalomma, Rhipicephalus, Dermacentor, Haemaphysalis and Ixodes were tested for the presence of CCHFV. Among 4,188 tick pools studied, the virus was detected in 252 (6%). The main vector, as expected, is H. marginatum (81% of positive pools). The same species is the most numerous among the studied ticks (66%). Apart from H. marginatum, the virus was detected in the following species: H. scupense, R. rossicus, R. turanicus, R. bursa, R. annulatus, D. marginatus, Haem. punctata and Ixodes ricinus. Most of the positive results were obtained from ticks collected from vertebrate hosts. As for H. marginatum, R. rossicus, D. marginatus, and Haem. punctata, the virus was detected in questing unfed specimens collected from vegetation and soil surface, which indicates the participation of these ticks in the circulation of the virus.     

Molecular discrimination of Hyalomma tick species serving as reservoirs and vectors for Crimean-Congo hemorrhagic fever virus in sub-Saharan Africa

The species identification of tick vectors of Crimean-Congo hemorrhagic fever virus (CCHFV), especially Hyalomma (H.) species, is a prerequisite to understand the eco-epidemiology of this disease and to reveal vector and virus reservoir species. However, the morphologic species discrimination can be difficult for damaged or blood-fed ticks and in case of species intercrosses. Therefore, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and restriction fragment length polymorphism (RFLP) analysis to distinguish the most common Hyalomma species from sub-Saharan Africa (H. truncatum, H. rufipes and H. dromedarii). Within the last years, MALDI-TOF MS analysis based on tick leg proteins has been shown to be a reliable method to distinguish several tick species. For this purpose, a reference spectral library of several European, American and African tick species was established. In this study, six different Hyalomma species were tested, all of which were all clearly distinguishable by mass spectrometric analyses. Moreover, MALDI TOF- MS was able to confirm morphologic findings where sequencing provided ambiguous results. In addition, a polymerase chain reaction (PCR) based on the CO1 gene amplification of ticks has been developed for the unequivocal species identification by amplicon sequencing and specific restriction endonuclease cleavage pattern analysis. RFLP proved to be a feasible auxiliary discrimination tool for selected Hyalomma species when access to sequencing methods is not available, as for instance during field studies.     

Genetic characterization and molecular clock analyses of the Crimean-Congo hemorrhagic fever virus from human and ticks in India, 2010–2011

A nosocomial outbreak of Crimean Congo hemorrhagic fever (CCHF) was reported among humans in Ahmadabad district, Gujarat, India during January, 2011. In the present study we provide the complete genomic sequences of four CCHFV isolates derived from two human patients and two pools of Hyalomma anatolicum ticks during the period of this outbreak and the complete S segment sequence of two retrospective human serum samples, positive for CCHFV in 2010. Sequence-based molecular characterization of the Indian CCHFV showed that they possessed the functional motifs known to occur in the S, M and L gene segment products as in other CCHF viruses. The S segment of the six Indian CCHF viruses showed 99.8% nucleotide identity. Notably both tick isolates shared 100% nucleotide identity with one of the Indian human isolates of 2011. Phylogenetic analysis based on the S segment demonstrated that the Indian CCHFV isolates formed a distinct cluster in the Asian-Middle East group IV of CCHF viruses. The S segment was closest to a Tajikistan strain TADJ/HU8966 of 1990 (98.5% nucleotide identity) and was of South-Asia 2 type while the M segment was of type M2. Both M and L segments were closest to an Afghanistan strain Afg09-2990 of 2009 (93% and 98% nucleotide identity, respectively). The Indian isolates were thus identified as a South-Asia 2/M2 far-east virus combination and the differing parental origin in the S and L/M segments is suggestive that it may be an intra-genotypic reassortant. Molecular clock studies further revealed that the ancestry of the viruses was not very recent and dated back to about 33 years on the basis of the S segment while it was about 15 years based on the M segment. Thus though the 2011 outbreak may not have resulted from a very recent introduction, considering that so far there is no evidence of multiple circulating strains in the country, the possibility of a recent re-introduction of the virus from any of the neighboring countries cannot be ruled out. The study thus warrants the need for continued surveillance and increased sampling of CCHFV in different parts of the country.     

A new report of adult Hyalomma marginatum and Hyalomma rufipes in the Czech Republic

Hyalomma marginatum and Hyalomma rufipes are important vectors of Crimean-Congo Hemorrhagic Fever Virus (CCHFV) in North Africa and Southern Europe. They are occasionally also reported from Central and Western Europe where they are likely introduced from their natural range by migratory birds. In this study, we report findings and molecular identification of adults and one nymph of H. marginatum and H. rufipes, primarily from horses from different regions of the Czech Republic. While the number of the reported ticks is small, this is likely to be an underrepresentation of the actual number. Due to their vector competence for CCHFV and potential expansion into new areas with a changing climate, surveillance programs in Europe are warranted.     

Development of vaccines against Crimean-Congo haemorrhagic fever virus

Crimean-Congo haemorrhagic fever virus (CCHFV) is a deadly human pathogen of the utmost seriousness being highly lethal causing devastating disease symptoms that result in intense and prolonged suffering to those infected. During the past 40 years, this virus has repeatedly caused sporadic outbreaks responsible for relatively low numbers of human casualties, but with an alarming fatality rate of up to 80% in clinically infected patients. CCHFV is transmitted to humans by Hyalomma ticks and contact with the blood of viremic livestock, additionally cases of human-to-human transmission are not uncommon in nosocomial settings. The incidence of CCHF closely matches the geographical range of permissive ticks, which are widespread throughout Africa, Asia, the Middle East and Europe. As such, CCHFV is the most widespread tick-borne virus on earth. It is a concern that recent data shows the geographic distribution of Hyalomma ticks is expanding. Migratory birds are also disseminating Hyalomma ticks into more northerly parts of Europe thus potentially exposing naïve human populations to CCHFV. The virus has been imported into the UK on two occasions in the last five years with the first fatal case being confirmed in 2012. A licensed vaccine to CCHF is not available. In this review, we discuss the background and complications surrounding this limitation and examine the current status and recent advances in the development of vaccines against CCHFV.     

河南省新疆出血热血清流行病学调查

目的调查河南省新疆出血热的感染情况.方法采集不同地区牧羊人、一般农民、发热病人、既往EHF患者及家属的人血清标本及动物血清标本作横断面调查,采用基于昆虫细胞表达的CCHFV核蛋白建立的ELISA法对血清标本进行初筛,阳性及可疑阳性再用表达CCHFV核蛋白的HeLa细胞进行IFA试验加以确证.结果254份人血清及242份羊血清标本全部为阴性.结论调查表明河南省尚未发现新疆出血热病毒感染.应进一步调查,加强监测,做好防范新疆出血热的准备,尤其预防新疆出血热的输入感染.     

The Saharan antelope addax (Addax nasomaculatus) as a host for Hyalomma marginatum,tick vector of Crimean-Congo hemorrhagic fever virus

Tick infestation and pathogen prevalence in ticks infesting the Saharan antelope addax (Addax nasomaculatus) are factors that may constitute a risk for both human and animal health. In this study we describe season distribution of adult Hyalomma marginatum and analyzed the tick-borne pathogens and their seroprevalence in natural-living addax in Morocco. The results showed that addax is an important host species for H. marginatum adults. The seroprevalence of Bluetongue virus (BTV; 61.5–92.3%, n = 8/13-84/91), Coxiella burnetii (36.3–69.2%, n = 33/91-9/13) and Brucella spp. (0.0–4.8%, n = 0/50-2/42) was characterized in addax during various years (sampled animals per year, n = 13–91). Presence of Aigai virus (AIGV), a recent taxonomic differentiation of Crimean-Congo hemorrhagic fever virus (CCHFV) of 100% (4/4, years 2016 and 2017) together with Babesia ovis (75%, 3/4, year 2014), Anaplasma spp. (75%, 3/4, year 2014), Rickettsia spp. (50%, 2/4, year 2014) and Theileria spp. (25%, 1/4, year 2014) was observed in H. marginatum collected from the addax (4 pools of 10 adult ticks each). The results support the role of addax host in H. marginatum life cycle and exposure to AIGV and other tick-borne pathogens. The development of control interventions including anti-tick vaccines for wildlife species will contribute to the implementation of effective measures for the prevention and control of tick-borne diseases and might be relevant for the preservation of this threatened species and others such as Arabian oryx (Oryx leucoryx) and African elk (Taurotragus oryx) that share habitat.     

Genetic characterization of the M RNA segment of Crimean Congo hemorrhagic fever virus strains, China.

We report the genetic characterization of the M RNA segment of Crimean Congo hemorrhagic fever virus (CCHFV). Two CCHFV strains isolated in Xinjiang Province, a region endemic for CCHF in northwestern China, were studied. These strains, designated BA66019 and BA8402, were isolated in 1965 and 1984 from a CCHF patient and Hyalomma ticks, respectively. Viral RNA was extracted from suckling mouse brains infected with these two strains, amplified, and sequenced. The full-length M RNA, consisting of 5.3 kb, was determined for both strains. The coding nucleotide sequences of the two strains differed from each other by 17.5% and from the reference CCHFV strain IbAr10200 by a mean of 22%, suggesting that the genus Nairovirus comprises a group of genetically highly diverse strains.     

The role of microRNAs in modulating SARS-CoV-2 infection in human cells: a systematic review

MicroRNAs are gene expression regulators, associated with several human pathologies, including the ones caused by virus infections. Although their role in infection diseases is not completely known, they can exert double functions in the infected cell, by mediating the virus infection and/or regulating the immunity-related gene targets through complex networks of virus-host cell interactions. In this systematic review, the Pubmed, EMBASE, Scopus, Lilacs, Scielo, and EBSCO databases were searched for research articles published until October 22nd, 2020 that focused on describing the role, function, and/or association of miRNAs in SARS-CoV-2 human infection and COVID-19. Following the PRISMA 2009 protocol, 29 original research articles were selected. Most of the studies reported miRNA data based on the genome sequencing of SARS-CoV-2 isolates and computational prediction analysis. The latter predicted, by at least one independent study, 1266 host miRNAs to target the viral genome. Thirteen miRNAs were identified by four independent studies to target SARS-CoV-2 specific genes, suggested to act by interfering with their cleavage and/or translation process. The studies selected also reported on viral and host miRNAs that targeted host genes, on the expression levels of miRNAs in biological specimens of COVID-19 patients, and on the impact of viral genome mutations on miRNA function. Also, miRNAs that regulate the expression levels of the ACE2 and TMPRSS2 proteins, which are critical for the virus entrance in the host cells, were reported. In conclusion, despite the limited number of studies identified, based on the search terms and eligibility criteria applied, this systematic review provides evidence on the impact of miRNAs on SARS-CoV-2 infection and COVID-19. Although most of the reported viral/host miRNAs interactions were based on in silico prediction analysis, they demonstrate the relevance of the viral/host miRNA interaction for viral activity and host responses. In addition, the identified studies highlight the potential use of miRNAs as therapeutic targets against COVID-19, and other viral human diseases (This review was registered at the International Prospective Register of Systematic Reviews (PROSPERO) database (#CRD42020199290).     

Phylogenetic analysis of Crimean-Congo hemorrhagic fever virus in inner Mongolia,China

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne arbovirus that can cause bleeding and death in humans. The mortality rate in humans is between 5 and 30%. The pathogen is prevalent in more than 30 countries in the world. In China, the CCHFV has been reported in Xinjiang province but not in Inner Mongolia province yet. In this report, we phylogenetically analyzed a new CCHFV strain, HANM-18, identified from Hyalomma asiaticum and Hyalomma dromedarii collected in Alxa Left Banner and Alxa Right Banner of Inner Mongolia, China. Complete sequences of CCHFV were obtained by the nested PCR technique and used for phylogenetic analysis of the identity and evolutionary relationship with other CCHFV strains. Our results showed that the S and L fragments of the HANM-18 strain had a high percentage of identity with strains in Xinjiang, China. The M fragment was significantly homologous to South African isolates. In addition, these data also indicate that the HANM-18 strain may have been prevalent in northwestern Inner Mongolia for many years. This discovery will be helpful in CCHF prevention and control in Inner Mongolia, and it also adds new evidence to the epidemiology of CCHF in China.     

Healthy individuals' immune response to the Bulgarian Crimean-Congo hemorrhagic fever virus vaccine

Crimean-Congo hemorrhagic fever virus (CCHFV) poses a great threat to public health due to its high mortality and transmission rate and wide geographical distribution. There is currently no specific antiviral therapy for CCHF. This study provides the first in-depth analysis of the cellular and humoral immune response in healthy individuals following injection of inactivated Bulgarian vaccine, the only CCHFV vaccine available at present. Vaccinated individuals developed robust, anti-CCHFV-specific T-cell activity as measured by IFN-γ ELISpot assay. The frequency of IFN-γ secreting T-cells was 10-fold higher in individuals after vaccination with four doses than after one single dose. High levels of CCHFV antibodies were observed following the first dose, but repeated doses were required to achieve antibodies with neutralizing activity against CCHFV. However, the neutralizing activity in these groups was low.     

Epidemiological investigations of Crimean-Congo haemorrhagic fever virus infection in sheep and goats in Balochistan,Pakistan

Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne zoonotic disease caused by the arbovirus Crimean-Congo haemorrhagic fever virus (CCHFV). Livestock serve as a transient reservoir for CCHFV, but do not show clinical signs. In this cross-sectional study, sheep and goats in Balochistan, Pakistan, were examined to determine the CCHFV seroprevalence, spatial distribution of seropositive sheep and goats, and to identify potential risk factors for seropositivity to CCHFV in these animals. To this end, farms and animals were selected by systematic sampling, blood samples from 800 sheep and 800 goats were collected and information regarding farm management and the kept animals were retrieved using a standard questionnaire. Sera were tested for antibodies against CCHFV in two independent ELISA formats and an immunofluorescence assay (IFA) following a hierarchical diagnostic decision tree. By these assays 149 (19 %, 95 %-CI: 16–21 %) out of 800 sheep serum samples and 37 (5 %, 95 %-CI: 3–6 %) out of 800 goat serum samples were positive for CCHFV-specific IgG antibodies. Interestingly, at least 8 (5 %, 95 %-CI: 2–10 %) out of 160 sera pools were from CCHFV viraemic sheep, as sera (in pools of 5) tested positive for CCHFV genome by real-time PCR (RT-qPCR). Risk factor analysis revealed that the open type of housing (OR = 3.76, 95 %-CI:1.57-9.56, p-value = 0.003), grazing (OR = 4.18, 95 %-CI:1.79-10.37, p-value = 0.001), presence of vegetation in or around the farm (OR = 3.13, 95 %-CI: 1.07–10.15, p-value = 0.043), lack of treatment against ticks (OR = 3.31, 95 %-CI: 1.16–10.21, p-value = 0.029), absence of rural poultry (OR = 2.93, 95 %-CI: 1.41–6.29, p-value = 0.004), animals with age ≥ 2 years (OR = 4.15, 95 %-CI: 2.84–6.19, p-value<0.001), animals infested with ticks (OR = 2.35, 95 %-CI: 1.59–3.52, p-value<0.001), and sheep species (OR = 4.72, 95 %-CI:3.24-6.86, p-value<0.001) represented statistically significant risk factors associated with seropositivity to CCHFV. Taken together this study confirms the circulation of CCHFV in livestock in Balochistan, Pakistan. The identification of risk factors might help to reduce the risk of infection in sheep and goats, which may also mitigate the risk for human infection. An interesting option for reducing the risk of CCHFV infection in small ruminants is keeping also chickens, since they pick ticks that transmit CCHFV.     

Vaccine efficacy trials for Crimean-Congo haemorrhagic fever: Insights from modelling different epidemiological settings

BackgroundCrimean-Congo haemorrhagic fever (CCHF) is a priority emerging pathogen for which a licensed vaccine is not yet available. We aim to assess the feasibility of conducting phase III vaccine efficacy trials and the role of varying transmission dynamics.MethodsWe calibrate models of CCHF virus (CCHFV) transmission among livestock and spillover to humans in endemic areas in Afghanistan, Turkey and South Africa. We propose an individual randomised controlled trial targeted to high-risk population, and use the calibrated models to simulate trial cohorts to estimate the minimum necessary number of cases (trial endpoints) to analyse a vaccine with a minimum efficacy of 60%, under different conditions of sample size and follow-up time in the three selected settings.ResultsA mean follow-up of 160,000 person-month (75,000–550,000) would be necessary to accrue the required 150 trial endpoints for a target vaccine efficacy of 60 % and clinically defined endpoint, in a setting like Herat, Afghanistan. For Turkey, the same would be achieved with a mean follow-up of 175,000 person-month (50,000–350,000). The results suggest that for South Africa the low endemic transmission levels will not permit achieving the necessary conditions for conducting this trial within a realistic follow-up time. In the scenario of CCHFV vaccine trial designed to capture infection as opposed to clinical case as a trial endpoint, the required person-months is reduced by 70 % to 80 % in Afghanistan and Turkey, and in South Africa, a trial becomes feasible for a large number of person-months of follow-up (>600,000). Increased expected vaccine efficacy > 60 % will reduce the required number of trial endpoints and thus the sample size and follow-time in phase III trials.ConclusionsUnderlying endemic transmission levels will play a central role in defining the feasibility of phase III vaccine efficacy trials. Endemic settings in Afghanistan and Turkey offer conditions under which such studies could feasibly be conducted.     

Crimean-Congo hemorrhagic fever in Turkey

In 2002 and 2003, a total of 19 persons in Turkey had suspected cases of Crimean-Congo hemorrhagic fever (CCHF) or a similar viral infection. Six serum samples were tested; all six were found positive for immunoglobulin M antibodies against CCHF virus. Two of the samples yielded CCHF virus isolates. Genetic analysis of the virus isolates showed them to be closely related to isolates from former Yugoslavia and southwestern Russia. These cases are the first of CCHF reported from Turkey. Eighteen patients handled livestock, and one was a nurse with probable nosocomial infection. The case-fatality rate was 20% among confirmed CCHF case-patients (1 of 5 patients), and the overall case-fatality rate was 11% (2 of 19 patients). In addition to previously reported symptoms and signs, we report hemophagocytosis in 50% of our patients, which is the first report of this clinical phenomenon associated with CCHF.     

miRNAs and genes expression in MARC-145 cell in response to PRRSV infection

The regulation of viral replication is under control of miRNAs and their target genes. Several articles report the cross-talk between host and virus. The drastic effects of Porcine reproductive and respiratory syndrome virus (PRRSV) pressed us to investigate the expression profiling of miRNAs and immunity related genes during PRRSV infection. This was performed by qPCR in MARC145 cells during PRRSV infection. It was observed that miRNAs and genes show different expression patterns at different time points during PRRSV infection. The early infected stage was accompanied with increased expression of some miRNAs including miR-204, miR-21, miR-181a, miR-29 while a decrease was observed for the same in late infection stage. The opposite condition also existed in parallel. An interesting observation was seen when miR-145 was strongly induced by PRRSV infection, whereas miR-127 expression was significantly reduced in all infection points. Taken together, our studies have revealed that the expressions of miRNAs and immune-related genes were regulated in PRRSV infected MARC-145 cells and had important roles in the immune response, providing a basis for further investigations.