Molecular detection of Crimean-Congo haemorrhagic fever virus in ticks collected from infested livestock populations in a New Endemic Area, South of Iran.

OBJECTIVE: Crimean-Congo haemorrhagic fever (CCHF) is a viral zoonotic disease with potentially fatal systemic effects on man. We aimed to determine the presence of CCHF virus among collected ticks from domestic livestock from October 2012 to September 2013. METHODS: A total of 1245 hard and soft ticks were collected from naturally infested ruminants in Marvdasht County, Fars Province, south of Iran. Nine tick species and one unidentified species in four disparate genera were detected. A total of 200 ticks were randomly selected and analysed by reverse transcription-polymerase chain reaction (RT-PCR) for the presence of CCHF virus genome. RESULTS: The viral genome was detected in 4.5% (9 samples) of the studied tick population. The infected ticks belonged to the species of Hyalomma marginatum' Hyalomma anatolicum and Rhipicephalus sanguineus. The viruses detected in these three tick species were clustered in the same lineage as Matin and SR3 strains in Pakistan and some other Iranian strains. These results indicate that the ticks were wildly infected with a genetically closely related CCHF virus in the region. CONCLUSION: Regular controls and monitoring of livestock to reduce the dispersion of ticks and providing information to those involved in high-risk occupations are urgently required.

Serological and molecular epidemiology of Crimean-Congo hemorrhagic fever in Ghaemshahr county in Mazandaran province; Iran.

Crimean-Congo Hemorrhagic Fever (CCHF) is a tick-borne viral hemorrhagic fever disease which is known as an endemic disease within some provinces of Iran. Ticks play an important role in transmission of the disease. As vector and reservoir, ticks transmit CCHF virus from livestock to human. The current study reports the presence of CCHFV in Ghaemshahr county of Mazandaran province, in north of Iran based on the evidences obtained from ELISA and RT- PCR. Based on our results, IgG antibodies against CCHFV were detected in 4(4.8%) out of 84 sheep sera samples. Forty sera were obtained from people who were in close contact with the examined sheep, none of which had IgG antibodies against CCHFV. Using RT-PCR, we confirmed the existence of CCHFV genome in 1.7% of hard tick samples. Sequence analysis demonstrated that CCHFV genomes isolated from ticks were 100% identical to those isolated from the corresponding livestock. This study confirms the presence of the virus in this region; so people in close contact with livestock and health care workers should be alerted.

Molecular diagnostic and genetic characterization of highly pathogenic viruses: application during Crimean-Congo haemorrhagic fever virus outbreaks in Eastern Europe and the Middle East.

Several haemorrhagic fevers are caused by highly pathogenic viruses that must be handled in Biosafety level 4 (BSL-4) containment. These zoonotic infections have an important impact on public health and the development of a rapid and differential diagnosis in case of outbreak in risk areas represents a critical priority. We have demonstrated the potential of a DNA resequencing microarray (PathogenID v2.0) for this purpose. The microarray was first validated in vitro using supernatants of cells infected with prototype strains from five different families of BSL-4 viruses (e.g. families Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae and Paramyxoviridae). RNA was amplified based on isothermal amplification by Phi29 polymerase before hybridization. We were able to detect and characterize Nipah virus and Crimean-Congo haemorrhagic fever virus (CCHFV) in the brains of experimentally infected animals. CCHFV was finally used as a paradigm for epidemics because of recent outbreaks in Turkey, Kosovo and Iran. Viral variants present in human sera were characterized by BLASTN analysis. Sensitivity was estimated to be 10(5) -10(6) PFU/mL of hybridized cDNA. Detection specificity was limited to viral sequences having ~13-14% of global divergence with the tiled sequence, or stretches of ~20 identical nucleotides. These results highlight the benefits of using the PathogenID v2.0 resequencing microarray to characterize geographical variants in the follow-up of haemorrhagic fever epidemics; to manage patients and protect communities; and in cases of bioterrorism.

Detection of West Nile virus genome and specific antibodies in Iranian encephalitis patients.

West Nile virus (WNV) is a mosquito-borne flavivirus which circulates in birds, horses and humans. An estimated 80% of WNV infections are asymptomatic. Fewer than 1% of infected persons develop neuroinvasive disease, which typically presents as encephalitis, meningitis, or acute flaccid paralysis. This study was conducted from January 2008 to June 2009 in Isfahan, Iran. Patients attending the emergency department with fever and loss of consciousness were consecutively included. Cerebrospinal fluids (CSF) were initially analysed through bacteriology and biochemistry examinations, resulting in those with evidence of meningitis being excluded. Patients' CSF and serum were diagnosed by serological and molecular assays. A total of 632 patients with fever and loss of consciousness were tested by CSF analyses. Samples of the remaining patients (39·4%) were referred for WNV investigation. Three (1·2%) of the patients were positive for both serum and CSF by RT-PCR, and six (2·4%) were positive only for IgG antibodies. History of insect bite, and blood transfusion and transplantation were risk factors for being positive by RT-PCR (P=0·048) and being IgG positive (P=0·024), respectively. The results of this study showed that the prevalence of West Nile fever is low in patients with encephalitis.

Seroepidemiology of crimean congo hemorrhagic Fever in slaughterhouse workers in north eastern iran.

BACKGROUND: Crimean Congo Hemorrhagic Fever (CCHF) is an acute febrile haemorrhagic disease. This study was conducted to ascertain the infection status amongst slaughterhouse workers in Iran's north-eastern provinces (Razavi and northern and southern Khorasan), so that analysis of the results could help clarification of the epidemiology of this disease in the aforementioned provinces and eastern regions of Iran. METHODS: In this cross-sectional study, conducted in 2004 and 2005, 108 slaughterhouse workers from 24 cities of the three previously mentioned provinces were randomly entered into the study. An IgG specific ELISA test was carried out on the participants' serum samples. RESULTS: Sixteen out of 108 (14.8%) participants under study were shown to have IgG against CCHF. The highest rate of infection was seen in Razavi Khorasan and southern Khorasan at 17.5% and 16.7%, respectively. CONCLUSION: The study showed a relatively high frequency of this disease amongst slaughterhouse workers in these provinces. Taking into account the small number of reported cases from these provinces, it would seem that more focus is required on primary diagnosis and on referral of suspected patients.

Mice orally immunized with a transgenic plant expressing the glycoprotein of Crimean-Congo hemorrhagic fever virus.

While Crimean-Congo hemorrhagic fever (CCHF) has a high mortality rate in humans, the associated virus (CCHFV) does not induce clinical symptoms in animals, but animals play an important role in disease transmission to humans. Our aim in this study was to examine the immunogenicity of the CCHFV glycoprotein when expressed in the root and leaf of transgenic plants via hairy roots and stable transformation of tobacco plants, respectively. After confirmatory analyses of transgenic plant lines and quantification of the expressed glycoprotein, mice were either fed with the transgenic leaves or roots, fed the transgenic plant material and injected subcutaneously with the plant-made CCHFV glycoprotein (fed/boosted), vaccinated with an attenuated CCHF vaccine (positive control), or received no treatment (negative control). All immunized groups had a consistent rise in anti-glycoprotein IgG and IgA antibodies in their serum and feces, respectively. The mice in the fed/boosted group showed a significant rise in specific IgG antibodies after a single boost. Our results imply that oral immunization of animals with edible materials from transgenic plants is feasible, and further assessments are under way. In addition, while the study of CCHF is challenging, our protocol should be further used to study CCHFV infection in the knockout mouse model and virus neutralization assays in biosafety level 4 laboratories.

Molecular epidemiology of Crimean- Congo hemorrhagic fever virus genome isolated from ticks of Hamadan province of Iran.

BACKGROUND & OBJECTIVES: Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne member of the genus Nairovirus, family Bunyaviridae. CCHFV has been isolated from at least 31 different tick species. The virus is transmitted through the bite of an infected tick, or by direct contact with CCHFV-infected patients or the products of infected livestock. This study was undertaken to study the genetic relationship and distribution of CCHFV in the tick population of Hamadan province of Iran. METHOD: In this study, RT-PCR has been used for detection of the CCHFV genome. RESULTS: This genome was detected in 19.2% of the ticks collected from livestock of different regions of the Hamadan province in western Iran. The infected species belonged to Hyalomma detritum, H. anatolicum, Rhipicephalus sanguineus and Argas reflexus. With one exception, genetic analysis of the virus genome isolates showed high sequence identity to each other. Even though they clustered in the same group with the strain circulating in Iran, they had a closer relationship to the Matin strain. INTERPRETATION & CONCLUSION: Vector control programs should be applied for reducing population density of potential tick vectors in this province. Further surveys are indicated in this region to provide a better view of the distribution and epidemiology of the virus.

Phylogenetic analysis in a recent controlled outbreak of Crimean-Congo haemorrhagic fever in the south of Iran, December 2008.

Crimean-Congo haemorrhagic fever (CCHF) is a viral zoonotic disease with a high mortality rate in humans. The CCHF virus is transmitted to humans through the bite of Ixodid ticks or contact with blood or tissues of CCHF patients or infected livestock. In December 2008, a re-emerging outbreak of CCHF occurred in the southern part of Iran. Five people were hospitalised with sudden fever and haemorrhaging, and CCHF was confirmed by RT-PCR and serological assays. One of the cases had a fulminant course and died. Livestock was identified as the source of infection; all animals in the incriminated herd were serologically analysed and more than half of them were positive for CCHFV. We demonstrated that two routes of transmission played a role in this outbreak: contact with tissue and blood of infected livestock, and nosocomial transmission. Phylogenetic analyses helped to identify the origin of this transmission. This outbreak should be considered as a warning for the national CCHF surveillance system to avoid further outbreaks through robust prevention and control programmes.

Hard Ticks on Domestic Ruminants and their Seasonal Population Dynamics in Yazd Province, Iran.

BACKGROUND: Ticks are the main vectors for transmission of different pathogens to human and animals. This survey was performed to find out distribution of ticks, which infested the domestic ruminants in Yazd Province, central Iran during year 2008-2009. METHODS: A total number of 30 villages from both mountainous (20%) and plateau (80%) regions of the province were selected randomly. Ticks were colleted from the body of infested animals and transported to the laboratory of Medical Entomology, School of Public Health, Tehran University of Medical Sciences and then were identified to space level using valid identification key. RESULTS: A total of 583 hard ticks were collected. The ticks were classified into three genera and 7 species including: Hyalomma dromedarii (55.92%), Hy. marginatum (13.20%), Hy. anatolicum (9.78%), Hy. detritum (4.98%), Hy. asiaticum (3.94%), Rhipicephalus sanguineus (11.84%), and Dermacentor marginatus (0.34%). The highest seasonal activities occurred in summer. The prevalence of the Ixodidae ticks was more evident in plateaus area in Yazd Province. Among the hosts including: cow, goat, sheep and camel, the ticks that collected from camel was more prevalent. The ratio of male was more than female ticks. Hyalomma. dromedarii was the predominant tick species and accounted for 55.92% of the ticks. CONCLUSION: Some of the collected ticks may play an important role for transmission of vector borne disease to human; therefore, the results of this study will provide a clue for vectors of tick-borne diseases in the region for local authorities for implementation of disease control.

Tick infestation rate of sheep and their distribution in abdanan county, ilam province, iran, 2007-2008.

BACKGROUND: Ticks are hematophagous arthropod belonging to the Class of Arachnids. Ticks are also one of the major vectors of pathogens to animal and human. This study was conducted to determine tick infestation rate of sheep in Abdanan during 2007-2008. METHODS: Sampling was performed seasonally in 19 villages during spring 2007 until winter 2008. A total of 1095 sheep were selected and tested for tick infestation. After collection, all ticks were transported to laboratory of Medical Entomology and were identified with appropriate identification keys. RESULTS: Totally, 864 hard ticks were collected. The ticks were classified into two genera and 5 species including: Hyalomma marginatum (44.67%), Hy. anatolicum (43.17%), Hy.asiaticum (6.37%), Hy. dromedarii (5.55%), Heamaphysalis sulcata (0.24%). The highest seasonal activity was observed in spring (36.46 %) and the lowest seasonal was in winter (11.57%). The rate of tick frequency in mountainous region was 48.15% and it was 51.85% in plateau regions. In this study, tick infestation of sheep was 11.41%. CONCLUSION: Hy.marginatum has the more frequent density in the study area.

Crimean-Congo hemorrhagic fever in Iran and neighboring countries.

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic viral disease that is asymptomatic in infected livestock, but a serious threat to humans. Human infections begin with nonspecific febrile symptoms, but progress to a serious hemorrhagic syndrome with a case fatality rate of 2-50%. Although the causative virus is often transmitted by ticks, livestock-to-human and human-to-human transmissions also occur. The disease is one of the most widely distributed viral hemorrhagic fevers occurring in Africa, the Middle East, Asia, and some parts of Europe. In this study, we have focused on the CCHF situation in Iran and neighboring countries and provide evidence of over 5000 confirmed cases of CCHF in a single period/season.

Surveillance and laboratory detection system of Crimean-Congo haemorrhagic fever in Iran.

Crimean-Congo haemorrhagic fever (CCHF) is a viral zoonotic disease with a high mortality rate in humans. The CCHF virus (CCHFV) is transmitted to humans through the bite of Ixodid ticks or by contact with blood or tissues of infected livestock. In addition to zoonotic transmission, CCHFV can be spread from person to person and is one of the rare haemorrhagic fever viruses able to cause nosocomial outbreaks in hospitals. Crimean-Congo haemorrhagic fever is a public health problem in many regions of the world such as Eastern Europe, Asia, the Middle East and Africa. In addition to clinical symptoms, the diagnosis of CCHF is based on the use of serological tests for the detection of immunoglobulin M and immunoglobulin G antibodies and on the use of molecular tools such as RT-PCR. From 1970 to 1978, serological and epidemiological studies were performed in humans and in livestock of Iran. After two decades and observations of CCHF in some provinces of Iran, a CCHF surveillance and detection system was established in 1999, leading to a dramatically decreased mortality rate from 20% (year 2000) to 2% (year 2007).

Evaluation of a Crimean-Congo hemorrhagic fever virus recombinant antigen expressed by Semliki Forest suicide virus for IgM and IgG antibody detection in human and animal sera collected in Iran.

Crimean-Congo hemorrhagic fever virus (CCHFV) is transmitted to humans by ticks or by direct contact with infected blood. It causes severe, often fatal, hemorrhagic diseases in humans but infection in animals is asymptomatic. CCHFV can spread from person to person and has caused many nosocomial outbreaks. Because the virus is very pathogenic for humans it must be manipulated in a biosafety level 4 (BSL4) laboratory, rendering the production of antigen for serological diagnosis difficult. To replace the native antigen, we produced a recombinant nucleoprotein expressed in mammalian cells via the recombinant Semliki Forest alphavirus replicon and developed an indirect immunofluorescence assay (IFA) as well as an enzyme-linked immunosorbent assay (ELISA) by immunocapture to detect IgM and IgG in human and animal serum. Using these methods, we analyzed clinical samples from human patients and sera from domestic animals collected in Iran and we show that this novel antigen provides a novel, sensitive and specific tool for CCHF diagnosis.