CCHFV RNA Research Articles

Crimean-Congo haemorrhagic fever outbreak in Northern Senegal in 2022: Prevalence of the virus in livestock and ticks, associated risk factors and epidemiological implications.

Crimean-Congo haemorrhagic fever (CCHF) is a severe zoonotic arboviral disease that occurs widely in Eastern and Western Europe, Asia and Africa. The disease is becoming of growing public health importance in Senegal. However, analysis of tick infestation, CCHF virus (CCHFV) circulation extent and risk factors during ongoing outbreak are scarce. A thorough outbreak investigation was carried out during a CCHF outbreak in Podor (Northern Senegal) in August 2022. Ticks and blood samples were collected from animals (cattle, goats and sheep) randomly selected from confirmed CCHF human cases houses, neighbourhoods and surrounding villages. Blood samples were tested for CCHFV antibodies using a commercial enzyme-linked immunosorbent assay (ELISA) test. Tick samples were screened for CCHFV RNA by RT-PCR. Overall, tick infestation rate (TIR) and CCHFV seroprevalence of livestock were 52.12% (95% confidence interval (CI): 45.54%-58.64%) and 43.28% (95% CI: 36.33%-50.44%), respectively. The TIRs were 87.7% in cattle, 57.6% in sheep and 20.0% in goats. These rates were significantly associated with location, host species and tick control (p < 0.001) but not with animal age and sex (p > 0.7). CCHFV seroprevalence was 80.4% (95% CI: 67.57%-89.77%) in cattle, 35.4% (95% CI: 25.00%-47.01%) in sheep and 21.2% (95% CI: 12.11%-33.02%) in goats. Age, sex, location, animal host and presence of ticks were significantly associated to the presence of antibodies. The 950 ticks collected included among other species, Hyalomma impeltatum (48.84%) and H. rufipes (10.21%). Five pools of Hyalomma ssp. were found CCHFV RT-PCR positive. These infected ticks included 0.86% (4/464) of H. impeltatum collected on cattle and sheep and 1.03% (1/97) of H. rufipes collected on a sheep. To our knowledge, this is the first report on the extend of tick infestation and CCHFV infection in livestock during an outbreak in Senegal. The results highlight the risk of human infections and the importance of strengthening vector, animal and human surveillance as well as tick control measures in this area to prevent CCHF infections in humans.

Risk factors associated with Crimean-Congo hemorrhagic fever virus circulation among human, livestock and ticks in Mauritania through a one health retrospective study

BackgroundCrimean Congo hemorrhagic fever (CCHF) is endemic in Southern Mauritania where recurrent outbreaks have been constantly observed since the 1980’s. The present study is the first to assess CCHFV antibodies and RNA in humans.MethodsA retrospective study was conducted using 263 humans and 1380 domestic animals serum samples, and 282 tick specimens of Hyalomma genus collected from 54 settings in 12 provinces across Mauritania. Antibodies targeting CCHF viral nucleoprotein were detected in animal and human sera using double-antigen ELISA. CCHFV specific RNA was detected in human and animal sera as well as tick supernatants using a CCHFV real time RT-PCR kit. Individual characteristics of sampled hosts were collected at the same time and data were geo-referenced. Satellite data of several environmental and climatic factors, were downloaded from publicly available datasets, and combined with data on livestock mobility, animal and human density, road accessibility and individual characteristics to identify possible risk factors for CCHFV spatial distribution. To this end, multivariate logistic models were developed for each host category (human, small and large ruminants).ResultsThe overall CCHFV antibody prevalence was 11.8% [95% CI: 8.4–16.3] in humans (17.9% in 2020 and 5.4% in 2021; p = 0.0017) and 33.1% (95% CI: 30.1–36.3) in livestock. CCHFV-specific antibodies were detected in 91 (18.1%) out of 502 sheep, 43 (9.0%) out of 477 goats, 144 (90.5%) out of 161 dromedaries and 179 (74.6%) out of 240 cattle. CCHFV RNA was detected in only 2 (0.7%) sera out of 263 animals herders samples from Hodh El Gharbi province and in 32 (11.3%) out of 282 Hyalomma ticks. In humans as well as in animals, seropositivity was not associated with sex or age groups. The multivariate analysis determined the role of different environmental, climatic and anthropic factors in the spatial distribution of the disease with animal mobility and age being identified as risk factors.ConclusionResults of the present study demonstrate the potential risk of CCHF for human population in Mauritania primarily those living in rural areas in close vicinity with animals. Future studies should prioritize an integrative human and veterinary approach for better understanding and managing Crimean-Congo hemorrhagic fever.

An Evaluation of the Hitit Index in Differential Diagnosis of Crimean-Congo Hemorrhagic Fever in the Emergency Department.

Background and Objectives: Crimean-Congo Hemorrhagic Fever (CCHF) is a viral zoonotic infection, which is seen over a wide geographic area. The mortality rate is in inverse proportion to the ability of patients to access healthcare services. Therefore, early identification of patients is extremely important. The aim of this study was to test the sensitivity and specificity of the Hitit Index in the differentiation of CCHF cases at the time of presentation at the Emergency Department and to evaluate the agreement of this index with molecular (CCHFV RNA) and/or serological diagnostic tests (ELISA-CCHF IgM). Materials and Methods: The patients included were those who presented at the Emergency Department (ED) with the complaint of a tick bite or those identified as potential CCHF cases as a result of complaints and/or laboratory findings. For cases that met the study inclusion criteria, the Hitit Index score was calculated automatically from the parameters included in the index formula uploaded to the automation system in the ED at the time of presentation. Through comparisons of the agreement of the Hitit Index with the CCHFV-RNA and/or IgM results the power of the Hitit Index for differentiation of CCHF cases in ED was evaluated. Results: The data of 273 patients were analyzed. There was a history of tick bite in 236 (86%) cases. Of the evaluated cases, 110 (40.2%) were hospitalized; CCHF positivity was determined in 72 (26.4%). The Hitit Index values calculated in ED and at 24 h after hospitalization were determined to be significant in the prediction of the CCHF cases (p < 0.001, AUC = 0.919 (0.887-0.951); p < 0.001, AUC = 0.902 (0.841-0.962). For a cut-off point of 0 of the Hitit Index evaluated in ED, the classification success was found to have a sensitivity of 75% and specificity of 88% (PPV-NPV). For a cut-off point of 0 of the Hitit Index evaluated at 24 h after hospitalization, the classification success was found to have a sensitivity of 79.7% and specificity of 84% (PPV-NPV). Conclusions: The defined form of the Hitit Index can be used in the differentiation of CCHF cases in ED with high sensitivity and specificity levels. Just as evaluation with the Hitit Index prevents unnecessary hospitalization, it can also contribute to reducing mortality rates with the early identification of CCHF cases.

Serologic and molecular evidence for circulation of Crimean-Congo hemorrhagic fever virus in ticks and cattle in Bosnia and Herzegovina

Crimean-Congo haemorrhagic fever virus is a tick-borne zoonotic agent of the genus Orthonairovirus, and the causal agent of Crimean-Congo haemorrhagic fever in humans. The study is aimed at investigating the presence of CCHFV RNA in ticks and cattle in Bosnia and Herzegovina (B&H), as well as the exposure of cattle to CCHFV in this country. Only one pool of three male Hyalomma marginatum ticks tested positive for viral RNA (3/760, 0.39%). No viral RNA was detected in 206 bovine blood samples using real-time reverse-transcriptase PCR (RT-qPCR). The overall seroprevalence of CCHFV in the cattle population was estimated as 14.97% (72/481). The results indicate the presence and the established circulation of CCHFV in B&H. Considering the endemic character of CCHFV in the Balkans as well as our results, the possibility that CCHFV is endemic in the territory of Bosnia and Herzegovina is considered to be very high.

Seroprevalence of Crimean-Congo Hemorrhagic Fever Among Small Ruminants from Southern Romania.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease that can be contracted by direct contact with viremic animals or humans. Domestic animals are accidental hosts and contribute to the spread and amplification of the virus. The main objective of this study was to provide updated information related to CCHF virus (CCHFV) infection in Southern Romania by assessing the seroprevalence of CCHF in small ruminants (sheep and goats) using a double-antigen sandwich enzyme-linked immunosorbent assay and by detection of CCHFV in engorged ticks and serum samples using real-time RT-PCR. The overall seroprevalence of CCHF in small ruminants was 37.7% (95% CI 31.7 to 43.7). No statistical seroprevalence difference was observed between the two species of ruminants (p = 0.76), but a significant difference was established between the locations (p < 0.01). No CCHFV RNA was detected in tick pools and small ruminant's sera tested by real-time RT-PCR, although the high seroprevalence to CCHFV among ruminants indicates that CCHV or a closely related virus circulates in Southern Romania.

Prevalence of Crimean-Congo Hemorrhagic Fever Virus among Livestock and Ticks in Zhambyl Region, Kazakhstan, 2017.

Crimean-Congo hemorrhagic fever (CCHF) is a highly fatal zoonotic disease endemic to Kazakhstan. Previous work estimated the seroprevalence of CCHF virus (CCHFV) among livestock owners in the Zhambyl region of southern Kazakhstan at 1.2%. To estimate CCHFV seroprevalence among cattle and sheep, we selected 15 villages with known history of CCHFV circulation (endemic) and 15 villages without known circulation (nonendemic) by cluster sampling with probability proportional to livestock population size. We collected whole blood samples from 521 sheep and 454 cattle from randomly selected households within each village and collected ticks found on the animals. We tested livestock blood for CCHFV-specific IgG antibodies by ELISA; ticks were screened for CCHFV RNA by real-time reverse transcription polymerase chain reaction and CCHFV antigen by antigen-capture ELISA. We administered questionnaires covering animal demographics and livestock herd characteristics to an adult in each selected household. Overall weighted seroprevalence was 5.7% (95% CI: 3.1, 10.3) among sheep and 22.5% (95% CI: 15.8, 31.2) among cattle. CCHFV-positive tick pools were found on two sheep (2.4%, 95% CI: 0.6, 9.5) and three cattle (3.8%, 95% CI: 1.2, 11.5); three CCHFV-positive tick pools were found in nonendemic villages. Endemic villages reported higher seroprevalence among sheep (15.5% versus 2.8%, P < 0.001) but not cattle (25.9% versus 20.1%, P = 0.42). Findings suggest that the current village classification scheme may not reflect the geographic distribution of CCHFV in Zhambyl and underscore that public health measures must address the risk of CCHF even in areas without a known history of circulation.

Risk factors associated with exposure to Crimean-Congo haemorrhagic fever virus in animal workers and cattle, and molecular detection in ticks, South Africa.

Crimean-Congo haemorrhagic fever (CCHF) is a severe tick-borne viral zoonosis endemic to parts of Africa, Europe, the Middle East and Central Asia. Human cases are reported annually in South Africa, with a 25% case fatality rate since the first case was recognized in 1981. We investigated CCHF virus (CCHFV) seroprevalence and risk factors associated with infection in cattle and humans, and the presence of CCHFV in Hyalomma spp. ticks in central South Africa in 2017–18. CCHFV IgG seroprevalence was 74.2% (95%CI: 64.2–82.1%) in 700 cattle and 3.9% (95%CI: 2.6–5.8%) in 541 farm and wildlife workers. No veterinary personnel (117) or abattoir workers (382) were seropositive. The prevalence of CCHFV RNA was significantly higher in Hyalomma truncatum (1.6%) than in H. rufipes (0.2%) (P = 0.002). Seroprevalence in cattle increased with age and was greater in animals on which ticks were found. Seroprevalence in cattle also showed significant geographic variation. Seroprevalence in humans increased with age and was greater in workers who handled livestock for injection and collection of samples. Our findings support previous evidence of widespread high CCHFV seroprevalence in cattle and show significant occupational exposure amongst farm and wildlife workers. Our seroprevalence estimate suggests that CCHFV infections are five times more frequent than the 215 confirmed CCHF cases diagnosed in South Africa in the last four decades (1981–2019). With many cases undiagnosed, the potential seriousness of CCHF in people, and the lack of an effective vaccine or treatment, there is a need to improve public health awareness, prevention and disease control.

Genetic diversity and phylogenetic analysis of Crimean Congo hemorrhagic fever virus in Pakistan

Background: Being an endemic country for Crimean Congo hemorrhagic fever (CCHF), this study aimed to explore the genetic diversity of CCHF virus (CCHFV) detected in Pakistan. This study was conducted to confirm the etiology of suspected hemorrhagic fever cases in order to support their clinical management. The genotype classification of CCHFV strains was determined to explore their phylogenetic relationship with strains in the region. Methods and materials: Serum samples from patients with clinical signs of hemorrhagic fever attending tertiary care hospitals in Pakistan, were tested for CCHFV RNA using real-time PCR at the Department of Virology, NIH. The partial S-gene fragments were directly sequenced to determine the prevailing CCHFV genotypes and their molecular epidemiology in Pakistan. Results: During Jan–Sep 2019, 178 samples from suspected CCHF patients were tested and 14 (8%) were found positive on real-time PCR. Phylogenetic analysis showed that all 2019 strains (n = 6) belonged to Asia-1 genotype and clustered with regional strains from Oman, Iran, UAE and Afghanistan. Fourteen CCHFV samples from 2013 outbreak were also retrospectively sequenced. Among 27 positive cases, phylogenetic analysis based on partial S-gene sequences of 15 CCHFV strains revealed that 73% strains (n = 11) belonged to Asia-1 genotype and clustered with viruses from Afghanistan and Iran. Three viruses were identified as Asia-2 genotype and grouped with isolates from India and Central Asian countries. Notably, one strain CCHF2013-56-NIHPAK was reported as Euro-1 genotype and clustered with isolates from Iran and Russia. Genetic diversity estimation of Pakistan strains (Aisa-1 genotype) detected during 2013 and 2019 showed 97–99% nucleotide homology. To our knowledge, this the first report on the detection of Euro-1 genotype from Pakistan Conclusion: We conclude that Asia-1 genotype of CCHF virus remains dominant in Pakistan over a period of seven years. Our findings emphasize establishing a laboratory-based surveillance program to monitor the disease burden and identify outbreak hotspots for effective control.

Genetic diversity and phylogenetic analysis of Crimean-Congo Hemorrhagic Fever viruses circulating in Pakistan during 2019.

Being an endemic country for Crimean Congo hemorrhagic fever (CCHF), this study aimed to explore the genetic diversity of CCHF virus (CCHFV) detected in Pakistan during 2019. Serum samples from patients with clinical signs of hemorrhagic fever attending tertiary care hospitals in Pakistan were tested for CCHFV RNA using real-time PCR at Department of Virology, National Institute of Health. The partial S-gene fragments were directly sequenced to determine the prevailing CCHFV genotypes and their molecular epidemiology in Pakistan. During January-December 2019, 280 samples from suspected CCHF patients were tested and 28 (10%) were found positive on real-time PCR. Positive cases were detected from 14 districts and across all four provinces of Pakistan with majority reported during August-September. The mean age of CCHFV positive patients was 37.25 years (range 5–65 years) with a high frequency in males (92.8%; n = 26) and a case fatality rate of 40.7% was observed. Phylogenetic analysis showed that S- segment of 2019 PAK CCHFV strains (n = 13) belonged to Asia-1 genotype and clustered with regional strains from Iran, Oman, and Afghanistan. We conclude that Asia-1 genotype of CCHF virus remains endemic in Pakistan. Our findings emphasize to establish a laboratory based surveillance program to monitor the disease burden and identify outbreak hotspots for effective control.

Results of investigation of ticks in Volga river delta (Astrakhan region, 2017) for Crimean-Congo hemorrhagic fever virus (Nairoviridae, Orthonairovirus, CCHFV) and other tick-borne arboviruses.

There are natural foci of Crimean-Congo hemorrhagic fever (CCHF) that vectored by Hyalomma marginatum ticks in Volga river delta (Astrakhan region, South of Russia). The circulation of Dhori virus (DHOV) (Thogotovirus: Orthomyxoviridae) has been also shown here. We hypothesized that other tick-borne arboviruses are also likely to circulate in the region. In particular, Bhanja virus (Phlebovirus: Phenuiviridae), Wad Medani virus (Orbivirus: Reoviridae), and Tamdy virus (Orthonairovirus: Nairoviridae), which were found to circulate in neighboring regions and are vectored by Haemaphysalis spp., Dermacenter spp., and Hyalomma spp. ticks. The aim of the study was to examine ixodid ticks in Volga river delta for the presence of CCHFV, DHOV, Bhanja virus, Wad Medani virus, and Tamdy virus. Ticks were collected in Volga river delta in 2017. We used molecular genetic methods for the detection and analysis of nucleic acids (PCR, sequencing, phylogenetic analysis). We detect CCHFV and DHOV RNA in H. marginatum ticks. The rate of infected H. marginatum ticks was 1.98% for CCHFV and 0.4% for DHOV. The results of genetic analysis showed that found DHOV strains are almost identical (99-100% in the M gene) and forms a separate genetic lineage alongside of Batken virus from Central Asia. At the same time, Bhanja virus, Wad Medani virus, and Tamdy virus were not found in ticks, collected in this region. DHOV is circulating in the natural foci of CCHF in the Volga river delta. The ratio of infection of H. marginatum with CCHFV and DHOV was determined for the first time.

Factors related to fatalities and clinical progression of Crimean-Congo hemorrhagic fever patients and the effects of IL 28-B gene polymorphism

Mortality rates of Crimean-Congo hemorrhagic fever (CCHF) vary from 5% to 80%. However, there is no clear information available about why this disease is fatal for some people while others recover. In this study, the factors related to fatalities and serious clinical progression of CCHF patients and the correlation between serious prognosis and IL 28-B gene polymorphism were investigated. The study included 107 patients with a preliminary diagnosis of CCHF, and the patients were found positive for CCHFV RNA based on polymerase chain reaction (PCR) analysis. The IL 28-B rs12979860 polymorphism was identified by PCR "restriction fragment length polymorphism" (PCR-RFLP) analysis using blood samples from the patients. In addition to the IL 28-B analysis results, a variety of data along with laboratory records obtained during the hospital stay were evaluated using statistical analysis. Of the 107 cases, nine were fatal (8.4%), while the other patients recovered and were discharged. Twenty-four patients had the CC genotype (22.43%), 64 had the CT genotype (59.81%), and 19 had the TT genotype (17.76%). Of the nine patients who died, three had the CC genotype (33.33%) and six had the CT genotype (66.67%). None of the patients who died had the TT genotype. Symptoms and findings of diarrhea, abdominal pain, hemorrhage, and rash were more common in fatal cases than in non-fatal cases. The IL 28-B rs12979860 polymorphism was not found to have a statistically significant correlation with fatality or symptoms indicating serious clinical progression in CCHF patients. As has been observed in previous studies, our study showed that leukocytosis, abdominal pain and diarrhea were more common in fatal cases.

Epidemiological Investigation of Crimean-Congo Hemorrhagic Fever infection in Cattle in some provinces of Turkey

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic disease which causes lethal hemorrhagic fever in humans. The purpose of this study was to investigate the CCHF infection status in cattle in the Central Anatolia region and the central-west part of the Aegean region of Turkey. For this purpose, EDTA whole blood (n = 329) and sera (n =329) samples were obtained from epidemiologically independent herds (n =94) during the months of March 2016 and September 2017. The exposure status to CCHF was determined using ELISA for detection of CCHF virus (CCHFV) specific IgG antibodies in cattle sera samples. Real-time reverse-transcriptase PCR was used to detect viral RNA in EDTA whole blood samples. The CCHFV-specific IgG antibodies were detected in 4 out of 329 animals, accounting for 1.2% prevalence rate. CCHFV RNA was not detected in EDTA whole blood samples. The low seroprevalence suggests only sporadic introduction of CCHFV. Further epidemiological studies are needed to determine the distribution of CCHFV infection in livestock in Turkey.

Investigation of an isolated case of human Crimean–Congo hemorrhagic fever in Central Uganda, 2015

BackgroundCrimean–Congo hemorrhagic fever (CCHF) is the most geographically widespread tick-borne viral infection. Outbreaks of CCHF in sub-Saharan Africa are largely undetected and thus under-reported. On November 9, 2015, the National Viral Hemorrhagic Fever Laboratory at the Uganda Virus Research Institute received an alert for a suspect VHF case in a 33-year-old male who presented with VHF compatible signs and symptoms at Mengo Hospital in Kampala. MethodsA blood sample from the suspect patient was tested by RT-PCR for CCHF and found positive. Serological testing on sequential blood specimens collected from this patient showed increasing anti-CCHFV IgM antibody titers, confirming recent infection. Repeat sampling of the confirmed case post recovery showed high titers for anti-CCHFV-specific IgG. An epidemiological outbreak investigation was initiated following the initial RT-PCR positive detection to identify any additional suspect cases. ResultsOnly a single acute case of CCHF was detected from this outbreak. No additional acute CCHF cases were identified following field investigations. Environmental investigations collected 53 tick samples, with only 1, a Boophilus decoloratus, having detectable CCHFV RNA by RT-PCR. Full-length genomic sequencing on a viral isolate from the index human case showed the virus to be related to the DRC (Africa 2) lineage. ConclusionsThis is the fourth confirmed CCHF outbreak in Uganda within 2 years after more than 50 years of no reported human CCHF cases in this country. Our investigations reaffirm the endemicity of CCHFV in Uganda, and show that exposure to ticks poses a significant risk for human infection. These findings also reflect the importance of having an established national VHF surveillance system and diagnostic capacity in a developing country like Uganda, in order to identify the first cases of VHF outbreaks and rapidly respond to reduce secondary cases. Additional efforts should focus on implementing effective tick control methods and investigating the circulation of CCHFV throughout the country.

Crimean-Congo hemorrhagic fever virus in ticks in Turkey: A broad range tick surveillance study

In Turkey, Crimean-Congo hemorrhagic fever (CCHF) outbreaks started in northern regions in 2002. Human cases still continue to increase and the disease spreads in many other provinces of Turkey. The ecological role of the vector tick species occurring in Turkey is not certain exactly. Therefore, we planned a broad range tick study in three different ecological and geographical areas extending from the West Black Sea regions down to the Central Anatolia. The aim of this study was to determine and characterize CCHFV molecularly in feeding-ticks collected from various wild and domestic animals and from humans as well as in questing and unfed ticks. In this study, 4283 tick samples derived from 21 tick species were collected from 76 villages and 10 central districts in total. All tick pools were screened for the presence of CCHFV RNA by two nested RT-PCRs. PCR assays were positive for 27 (3.6%) of 736 pools. CCHFV was detected in Hyalomma marginatum, Dermacentor marginatus, Rhipicephalus bursa, Rhipicephalus turanicus, Hyalomma excavatum and Haemaphysalis parva pools. As a result of the phylogenetic analysis, it was determined that the obtained CCHFV sequences are clustered into Europe 1 clade. CCHFV was detected among ticks obtained from cattle, goats, wild boar, hare, and the ground. The presence of CCHFV in ticks obtained from various domestic and wild hosts and the nature, and thereby the distribution patterns of the virus in different ecological areas were presented in this study. Furthermore, CCHFV was also detected in unusual tick species. Consequently, these results indicate that tick surveillance studies in large-scale and wide varieties contribute to the ecology and epidemiology of CCHF in that region, and can be used as an early-warning system.

EPIDEMIOLOGICAL SITUATION ON CRIMEAN HEMORRHAGIC FEVER IN THE RUSSIAN FEDERATION IN 2016, AND PROGNOSIS FOR 2017

Abstract. This paper presents analysis of epidemiological situation on Crimean hemorrhagic fever (CHF) in Russia in 2016. Summarized are the results of epizootiological survey of the territory of the natural CHF focus in the south of the European part of Russia, discussed are the results of genetic typing of CCHFV RNA isolates. In 2016, the Russian Federation reported 162 cases of CHF. Increase in the incidence of CHF occurred in the Astrakhan, Volgograd Regions, Republic of Kalmykia, and Stavropol Territory. For the first time CHF case was identified in Kabardino-Balkar Republic. In 2016 in stationary points for the long-term observation of the natural CHF focus, high abundance rates of larvae and nymphs of H. marginatum remain. In case of successful Ixodidae ticks wintering and late onset of the hot and dry season in the summer, 2017, there is probability that high numbers of H. marginatum will be retained and the period of the imago activity is prolonged, which may in its turn contribute to the increase in CHF morbidity rates.

Pseudo-plaque reduction neutralization test (PPRNT) for the measurement of neutralizing antibodies to Crimean-Congo hemorrhagic fever virus

BackgroundCrimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus family Bunyaviridae, which are enveloped viruses containing tripartite, negative polarity, single-stranded RNA. CCHF is characterized by high case mortality, occurring in Asia, Africa, the Middle East and Europe. Currently, there are no specific treatments or licensed vaccines available for CCHFV. Recently, two research groups have found adult mice with defective interferon responses allowed to lethal CCHFV infection. These mouse models could provide invaluable information for further studies. Efforts to develop a vaccine against CCHFV are being made. To determine the efficacy of vaccine candidates it is important to conduct serological studies that can accurately measure levels of protective antibodies. In the present study, a pseudo-plaque reduction neutralization test (PPRNT) based on enzyme-catalyzed color development of infected cells probed with anti-CCHFV antibodies was used to measure neutralization antibody of CCHFV.MethodsSixty-nine human serum samples (20 acute and 49 convalescent) were tested. The presence of CCHFV antibodies was determined and confirmed by a commercial ELISA kit. CCHFV RNA was determined by RT-PCR. All the samples were analyzed by PPRNT and fluorescent focus reduction neutralization test (FFRNT) to measure of CCHFV-neutralizing antibodies.ResultsPseudo-plaque reduction neutralization test showed a high sensitivity (98%), specificity (100%) and agreement (96,6%) in qualitative comparison with those of the FFRNT. There was a high correlation between the titers obtained in PPRNT and FFRNT (R2 = 0.92). The inter- and intra-assay variation of PPRNT revealed good reproducibility and positive cut-off of PPRNT was defined as 1:4 by the geometric mean titers for the individual samples distributed.ConclusionThe pseudo-plaque reduction neutralization test described in this study is a fast, reproducible and sensitive method for the measurement of CCHF neutralizing antibodies. This novel assay could serve as useful tools for CCHF research in epidemiology, vaccine development and other studies of immunity. It also provides an alternative to PRNT when viruses with no or poor CPE in cell culture.

Türkiye’deki Kırm Kongo Kanamalı Ateşi Virüslerinin Kısmi M-segmentlerinin Genetik Analizi

Crimean-Congo hemorrhagic fever (CCHF) is a fatal tick-borne zoonosis extensively common in Africa, Asia, Eastern Europe and the Balkan Peninsula. CCHF has been reported in Turkey with high frequency since 2002. Genetic diversity of CCHF virus (CCHFV) isolates circulating in Turkey were studied by two recent studies from 2006 to the end of 2010. Since CCHFV disease has been an important public health concern in Turkey, it is necessary to continue genetic analysis of CCHFV viruses for the assessment of future patterns of disease. The aim of the present study was to genetic analysis of CCHFV isolates derived from infected patients over a two-year period (2011 and 2012) in several provinces of Turkey. Serum samples (n=10) were selected from CCHFV RNA positive patients and subjected to sequence analysis of the gene region encoding partial M segment. The nucleotide sequence alignments of the 10 partial M segments of CCHFV isolates showed that the nucleic acid relatedness of CCHFV isolates ranged from 94.4% to 100%. Phylogenetic analysis of M segment sequences revealed that CCHFV isolates circulating in Turkey belonged to the European lineage I and were closely related to the viruses previously found in Turkey and in the Eastern European-Russian and Balkan Peninsula. The results of the present study indicated the genetic stability and the lack of the genetic diversity of CCHFV isolates circulating in Turkey.

First International External Quality Assessment of Molecular Detection of Crimean-Congo Hemorrhagic Fever Virus

Crimean-Congo hemorrhagic fever (CCHF) is a zoonosis caused by a Nairovirus of the family Bunyaviridae. Infection is transmitted to humans mostly by Hyalomma ticks and also by direct contact with the blood or tissues of infected humans or viremic livestock. Clinical features usually include a rapid progression characterized by hemorrhage, myalgia and fever, with a lethality rate up to 30%. CCHF is one of the most widely distributed viral hemorrhagic fevers and has been reported in Africa, the Middle East and Asia, as well as parts of Europe. There is no approved vaccine or specific treatment against CCHF virus (CCHFV) infections. In this context, an accurate diagnosis as well as a reliable surveillance of CCHFV infections is essential. Diagnostic techniques include virus culture, serology and molecular methods, which are now increasingly used. The European Network for the Diagnostics of “Imported” Viral Diseases organized the first international external quality assessment of CCHVF molecular diagnostics in 2011 to assess the efficiency and accurateness of CCHFV molecular methods applied by expert laboratories. A proficiency test panel of 15 samples was distributed to the participants including 10 different CCHFV preparations generated from infected cell cultures, a preparation of plasmid cloned with the nucleoprotein of CCHFV, two CCHFV RNA preparations and two negative controls. Forty-four laboratories worldwide participated in the EQA study and 53 data sets were received. Twenty data sets (38%) met all criteria with optimal performance, 10 (19%) with acceptable performance, while 23 (43%) reported results showing a need for improvement. Differences in performance depended on the method used, the type of strain tested, the concentration of the sample tested and the laboratory performing the test. These results indicate that there is still a need for improving testing conditions and standardizing protocols for the molecular detection of Crimean-Congo hemorrhagic fever virus.

Lack of genetic diversity in Crimean‐Congo hemorrhagic fever viruses in Turkey: Assessment of present and future patterns of disease

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis which is common in Africa, Asia, Eastern Europe, and the Balkan Peninsula. CCHF has been reported in Turkey with high frequency since 2002. The aim of the present study was to investigate the genetic diversity and genetic relationship between CCHF virus (CCHFV) isolates derived from infected patients over a 2-year period (2009 and 2010) in several provinces of Turkey. Serum samples (n = 48) were selected from CCHFV RNA positive patients and subjected to sequence analysis of the gene regions encoding the S (48 samples) and M (14 samples) segments. The nucleotide sequence alignments showed that the nucleic acid relatedness of CCHFV isolates ranged from 95.7% to 100% and from 93.7% to 100% for S and M segments, respectively. Phylogenetic analysis of both segment sequences revealed that CCHFV isolates circulating in Turkey belonged to the European lineage I and were closely related to the viruses found in the Eastern European-Russian and Balkan Peninsula. The M gene segment-based phylogenetic analysis suggested that 2/14 CCHFV isolates (KYSR3159/09 and YZGT714/10) had additional genetic variations. The results of the present study confirmed that the CCHFV isolates present in Turkey associated with human disease had high genetic homology in S segment, but some variability in the M segment of the RNA.

Investigation of the Presence of Crimean-Congo Hemorrhagic Fever Virus RNA in Tears of Eleven Infected Patients

Purpose: To investigate the presence of viral RNA in tears of patients with Crimean-Congo hemorrhagic fever (CCHF) disease. Methods: This study was conducted on eleven patients with CCHF cases. The diagnosis of CCHF was based on clinical and epidemiological findings and the detection of CCHF virus (CCHFV)-specific IgM and of CCHFV RNA in the serum samples of the patients by ELISA and a real time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. CCHFV RNA positive patients tear samples were then subjected for investigation of viral RNA. Blood and tears of patients were collected at the same time within 24 hours of hospitalization of patients. A TaqMan-based onestep real time RT-PCR was applied to detect CCHFV RNA in sera and tears of patients. Results: Eight patients were male and three patients were female. One of 11 patients had only CCHF virus-specific IgM antibodies and four of 11 patients' blood sera had only positive for viral RNA. Both viral RNA and virus specific IgM were detected in six out of eleven patients' sera. Although the presence of CCHFV RNA was confirmed in 10 (90.9%) patients' sera, none of the patients' tear had viral RNA. Conclusions: The preliminary results obtained in the present study showed that the presence of CCHFV RNA was not detected in tear fluids in patients having CCHF disease. This suggested that tears of CCHF patients may not have a potential for CCHFV virus transmission.