West Nile Virus lineage-2 in Culex specimens from Iran.

OBJECTIVE: Screening of mosquitoes for viruses is an important forecasting tool for emerging and re-emerging arboviruses. Iran has been known to harbour medically important arboviruses such as West Nile virus (WNV) and dengue virus (DENV) based on seroepidemiological data. However, there are no data about the potential mosquito vectors for arboviruses in Iran. This study was performed to provide mosquito and arbovirus data from Iran. MATERIALS AND METHODS: A total of 32 317 mosquitos were collected at 16 sites in five provinces of Iran in 2015 and 2016. RT-PCR for detection of flaviviruses was performed. The PCR amplicons were sequenced, and 109 WNV sequences, including one obtained in this study, were used for phylogenetic analyses. RESULTS: The 32 317 mosquito specimens belonging to 25 species were morphologically distinguished and distributed into 1222 pools. Culex pipiens s.l. comprised 56.429%. One mosquito pool (0.08%), containing 46 unfed Cx. pipiens pipiens form pipiens (Cpp) captured in August 2015, was positive for flavivirus RNA. Subsequent sequencing and phylogenetic analyses revealed that the detected Iranian WNV strain belongs to lineage 2 and clusters with a strain recently detected in humans. No flaviviruses other than WNV were detected in the mosquito pools. CONCLUSION: Cpp could be a vector for WNV in Iran. Our findings indicate recent circulation of WNV lineage-2 strain in Iran and provide a solid base for more targeted arbovirus surveillance programs.

Vector prevalence and detection of Crimean-Congo haemorrhagic fever virus in Golestan Province, Iran.

BACKGROUND & OBJECTIVES: Crimean-Congo haemorrhagic fever virus (CCHFV) causes severe disease with fatality rate of 30%. The virus is transmitted to humans through the bite of an infected tick, direct contact with the products of infected livestock as well as nosocomially. The disease occurs sporadically throughout many of African, Asian and European countries. Different species of ticks serve either as vector or reservoir for CCHFV. This study was aimed to determine the prevalence of CCHFV in hard ticks (Ixodidae) in the Golestan Province of Iran. METHODS: A molecular survey was conducted on hard ticks (Ixodidae) isolated from six counties in Golestan Province, north of Iran during 2014-15. The ticks were identified using morphological characteristics and presence of CCHFV RNA was detected using RT-PCR. RESULTS: Data revealed the presence of CCHFV in 5.3% of the ticks selected for screening. The infected ticks belonged to Hyalomma dromedarii, Hy. anatolicum, Hy. marginatum and Rhipicephalus sanguineus species. INTERPRETATION & CONCLUSION: The study demonstrated that Hyalomma ticks are the main vectors of CCHFV in Golestan Province. Thus, preventive strategies such as using acaricides and repellents in order to avoid contact with Hyalomma ticks are proposed.

Biosafety standards for working with Crimean-Congo hemorrhagic fever virus.

In countries from which Crimean-Congo haemorrhagic fever (CCHF) is absent, the causative virus, CCHF virus (CCHFV), is classified as a hazard group 4 agent and handled in containment level (CL)-4. In contrast, most endemic countries out of necessity have had to perform diagnostic tests under biosafety level (BSL)-2 or -3 conditions. In particular, Turkey and several of the Balkan countries have safely processed more than 100 000 samples over many years in BSL-2 laboratories. It is therefore advocated that biosafety requirements for CCHF diagnostic procedures should be revised, to allow the tests required to be performed under enhanced BSL-2 conditions with appropriate biosafety laboratory equipment and personal protective equipment used according to standardized protocols in the countries affected. Downgrading of CCHFV research work from CL-4, BSL-4 to CL-3, BSL-3 should also be considered.

First phylogenetic analysis of a Crimean-Congo hemorrhagic fever virus genome in naturally infected Rhipicephalus appendiculatus ticks (Acari: Ixodidae).

Crimean-Congo haemorrhagic fever (CCHF) is a potentially fatal systemic viral disease in many parts of the world, including Iran. The nationwide incidence of human CCHF in endemic areas was 870 confirmed cases with 126 deaths (case fatality rate, CFR = 17.6 %) in the decade leading to 2012. The detection of the CCHF virus (CCHFV) genome in tick vectors is of fundamental importance for identifying these ticks as potential reservoirs of CCHFV infection. From May to October 2013, following detection of four new clinical cases resulting in two deaths in the city of Mashhad (northeast Iran), hard ticks were recovered from infested livestock in 40 villages in Khorasan-Razavi province and examined by the microscopic method for species identification. About a quarter of the ticks were then subjected to reverse-transcription polymerase chain reaction (RT-PCR) to detect the CCHFV genome. The PCR products were then sequenced, and their phylogenetic lineages were determined. A total of 407 hard ticks were captured, representing seven different species in two distinct genera. Members of the genus Hyalomma were widely distributed in all but two of the villages studied, and this was also the most frequent (83.3 %) tick genus. Of 105 adult ticks subjected to RT-PCR, four (3.8 %) ticks were found positive for the CCHFV genome. One brown ear tick, Rhipicephalus appendiculatus, was found to be naturally infected for the first time anywhere in the world. Ticks of Hyalomma asiaticum, Hyalomma marginatum, and Rhipicephalus turanicus were also found to be naturally infected with CCHFV. CCHFV found in these four different tick species were clustered in the same lineage with the Matin and SR3 strains from Pakistan and some other strains from Iran, indicating that these tick species were naturally infected with genetically closely related CCHFV in the region. The presence of CCHFV infection in four different hard tick species was confirmed using RT-PCR in northeast Iran. Part of this infection was attributed to Rh. appendiculatus, which is thus a potential new natural vector of CCHFV in Iran. It is also confirmed by phylogenetic analysis that CCHFV in this region is genetically closely related, even in the different tick species.

Crimean-Congo Hemorrhagic Fever Virus Clade IV (Asia 1) in Ticks of Western Iran.

Crimean-Congo Hemorrhagic Fever virus (CCHFV) is transmitted through the bite of an infected tick, or by direct contact with CCHFV-infected patients' blood or the products of infected livestock. In 2012, ticks were collected in eight regions of Lorestan Province, Iran. In total, 434 ticks were collected. Reverse transcriptase polymerase chain reaction was used for the detection of CCHFV RNA. Of 434 ticks, 419 (96.6%) ticks were from the family Ixodidae (hard ticks) and 15 (3.5%) ticks were from the family Argasidae (soft ticks). The presence of CCHFV RNA was detected in 29 (6.7%) of 434 ticks. The infected tick species include Hyalomma asiaticum (n = 7, 7.4%), Hyalomma anatolicum (n = 12, 13.2%), Hyalomma marginatum (n = 1, 16.7%), and Rhipicephalus sanguineus (n = 9, 4.3%). These empirical data demonstrated that the majority of CCHFV-positive ticks belonged to the Ixodidae. None of the Argasidae and Haemaphysalis sulcata species was infected with CCHFV. The phylogenetic analyses of the tick-derived CCHFV strains revealed that all 29 viral strains fell in clade IV (Asia 1). The most abundant species of tick collected in this study was R. sanguineus followed by different species of Hyalomma. Given the infection rate among collected ticks, H. marginatum was the most abundant infected tick species (16.7%) followed by H. anatolicum (13.2%), H. asiaticum (7.4%), and R. sanguineus (4.3%).

Clinical symptoms and laboratory findings supporting early diagnosis of Crimean-Congo hemorrhagic fever in Iran.

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic disease, which is usually transmitted to humans by tick bites or contact with blood or other infected tissues of livestock. Patients suffering from CCHF demonstrate an extensive spectrum of clinical symptoms. As it can take considerable time from suspecting the disease in hospital until reaching a definitive diagnosis in the laboratory, understanding the clinical symptoms and laboratory findings of CCHF patients is of paramount importance for clinicians. The data were collected from patients who were referred to the Laboratory of Arboviruses and Viral Hemorrhagic Fevers at the Pasteur institute of Iran with a primary diagnosis of CCHF between 1999 and 2012 and were assessed by molecular and serologic tests. Referred patients were divided into two groups: patients with a CCHF positive result and patients with a CCHF negative result. The laboratory and clinical findings of these two groups were then compared. Two-thousand five hundred thirty-six probable cases of CCHF were referred to the laboratory, of which 871 cases (34.3%) were confirmed to be CCHF. Contact with infected humans and animals increased the CCHF infection risk (P < 0.001). A tick bite was not a risk factor. Fever; bleeding, vomiting, leucopoenia, thrombocytopenia, and increases in alanine transaminase (ALT) and aspartate transaminase (AST) levels were also indicative of CCHF infection. Accurate and speedy diagnosis of CCHF and appropriate treatment play an important role in patient survival and the application of the findings of this study can prove helpful as a key for early diagnosis.

Frequency of dengue virus infection in blood donors in Sistan and Baluchestan province in Iran.

Risk of dengue virus in the blood supply has been demonstrated in recent studies. In this paper, Chabahar in Sistan and Baluchestan province, south east of Iran, was selected for studying dengue infection because of its climatic and geographical situation in the middle of the transit way between East Asia and other countries. The blood samples were taken from volunteer healthy donors who were referred to the Chabahar Blood Center for blood donation. The presence of dengue virus (DENV) was studied by detecting IgG to DENV by enzyme linked immune sorbent assay (ELISA). Reactive ELISA results were confirmed by an immune flouorescence assay (IFA). According to the results, some of the healthy donors were infected by DENV, which could not been recognized in donor selection. Therefore, special attention should be paid to the criteria of donor selection and additional screening tests are recommended.

New circulating genomic variant of Crimean-Congo hemorrhagic fever virus in Iran.

Crimean-Congo hemorrhagic fever is a viral infection that is caused by Crimean-Congo hemorrhagic fever virus (CCHFV). On May 27, 2012, a woman became ill after accidentally splashing cow's blood into her eyes. Serological and molecular investigations were carried out on the serum of the patient. The test results for serological testing were negative, but RT-PCR was strongly positive for CCHFV. A phylogenetic study on the CCHFV genome sequence showed 50 % similarity to a 520-bp region of Russian strains. By combining historical phylogenetic data and current data, it can be surmised that there are potentially more than five circulating CCHFV genomic variants in Iran.

Diagnostic assays for Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever (CCHF) is a highly contagious viral tick-borne disease with case-fatality rates as high as 50%. We describe a collaborative evaluation of the characteristics, performance, and on-site applicability of serologic and molecular assays for diagnosis of CCHF. We evaluated ELISA, immunofluorescence, quantitative reverse transcription PCR, and low-density macroarray assays for detection of CCHF virus using precharacterized archived patient serum samples. Compared with results of local, in-house methods, test sensitivities were 87.8%-93.9% for IgM serology, 80.4%-86.1% for IgG serology, and 79.6%-83.3% for genome detection. Specificity was excellent for all assays; molecular test results were influenced by patient country of origin. Our findings demonstrate that well-characterized, reliable tools are available for CCHF diagnosis and surveillance. The on-site use of such assays by health laboratories would greatly diminish the time, costs, and risks posed by the handling, packaging, and shipping of highly infectious biologic material.

Colorimetric nucleic acid testing assay for RNA virus detection based on circle-to-circle amplification of padlock probes.

We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involves easier assay design, and has a sensitivity of 10(3) viral copies/ml. By using a cocktail of padlock probes, synthetic templates representing different viral strain variants could be detected. We analyzed 34 CCHF patient samples, and all patients were correctly diagnosed when the results were compared to those of the current real-time PCR method. This is the first time that highly specific padlock probes have been applied to detection of a highly variable target sequence typical of RNA viruses.

SARS-CoV-2 spillover transmission due to recombination event.

In late 2019, a novel Coronavirus emerged in China. Perceiving the modulating factors of cross-species virus transmission is critical to elucidate the nature of virus emergence. Using bioinformatics tools, we analyzed the mapping of the SARS-CoV-2 genome, modeling of protein structure, and analyze the evolutionary origin of SARS-CoV-2, as well as potential recombination events. Phylogenetic tree analysis shows that SARS-CoV-2 has the closest evolutionary relationship with Bat-SL-CoV-2 (RaTG13) at the scale of the complete virus genome, and less similarity to Pangolin-CoV. However, the Receptor Binding Domain (RBD) of SARS-CoV-2 is almost identical to Pangolin-CoV at the aa level, suggesting that spillover transmission probably occurred directly from pangolins, but not bats. Further recombination analysis revealed the pathway for spillover transmission from Bat-SL-CoV-2 and Pangolin-CoV. Here, we provide evidence for recombination event between Bat-SL-CoV-2 and Pangolin-CoV that resulted in the emergence of SARS-CoV-2. Nevertheless, the role of mutations should be noted as another influencing factor in the continuing evolution and resurgence of novel SARS-CoV-2 variants.

Crimean-Congo Hemorrhagic Fever Virus in Asia, Africa and Europe.

The global spread of ticks and various tick-borne viruses (TBVs) suggests the possibility of new tick-borne diseases emerging. Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging TBV of the Nairoviridae family that causes serious disease that can be fatal in humans. CCHFV endemic foci can be found in Africa, Asia, the Middle East, and South-Eastern Europe, and has spread to previously unaffected regions and nations, such as Spain, over the last two decades. In this review, we discuss the current situation of CCHFV in Asia, Africa and Europe based on existing knowledge, and we discuss driving factors in the distribution and transmission of the virus, such as the spread of tick vector species and host reservoirs.

A survey of Crimean-Congo haemorrhagic fever in livestock and ticks in Ardabil Province, Iran during 2004-2005.

Crimean-Congo haemorrhagic fever (CCHF) is a viral haemorrhagic fever caused by the CCHF virus. It is mainly transmitted to humans and animals by ticks. In recent y, large numbers of livestock have been transported across the border areas of Ardabil Province resulting in an outbreak of CCHF in the adjacent districts. A comprehensive study was carried out to assess the epidemiological aspects of the disease in this province. In the study area, 130 ticks were collected from randomly selected villages and classified into 9 species of hard tick and 2 species of soft tick. All ticks were analyzed for the presence of CCHF virus genome using gel-based and real-time reverse transcriptase polymerase chain reactions (RT-PCR). The results showed CCHF infection in almost 28% of ticks collectively. Also, of 56 livestock sera, around 39% were IgG-positive. The presence of anti-CCHF virus IgG antibodies and the CCHF virus genome in ticks points to a great hidden threat of an outbreak in these districts. Those in high-risk professions in this province should be informed and trained on the risk of CCHF with urgency.

Autochthonous Transmission of West Nile Virus by a New Vector in Iran, Vector-Host Interaction Modeling and Virulence Gene Determinants.

Using molecular techniques and bioinformatics tools, we studied the vector-host interactions and the molecular epidemiology of West Nile virus (WNV) in western Iran. Mosquitoes were collected during 2017 and 2018. DNA typing assays were used to study vector-host interactions. Mosquitoes were screened by RT-PCR for the genomes of five virus families. WNV-positive samples were fully sequenced and evolutionary tree and molecular architecture were constructed by Geneious software and SWISS-MODEL workspace, respectively. A total of 5028 mosquito specimens were collected and identified. The most prevalent species was Culex (Cx.) pipiens complex (57.3%). Analysis of the blood-feeding preferences of blood-fed mosquitoes revealed six mammalian and one bird species as hosts. One mosquito pool containing non-blood-fed Cx. theileri and one blood-fed Culex pipiens pipiens (Cpp.) biotype pipiens were positive for WNV. A phylogram indicated that the obtained WNV sequences belonged to lineage 2, subclade 2 g. Several amino acid substitutions suspected as virulence markers were observed in the Iranian WNV strains. The three-dimensional structural homology model of the E-protein identified hot spot domains known to facilitate virus invasion and neurotropism. The recent detection of WNV lineage 2 in mosquitoes from several regions of Iran in consecutive years suggests that the virus is established in the country.

Morphological and molecular identification of Culicidae mosquitoes (Diptera: Culicidae) in Lorestan province, Western Iran.

Culicidae mosquitoes are main vectors of arboviruses that cause arboviral diseases in humans. Studies on fauna, ecology, biology, resting behaviors of Culicidae mosquitoes are important and greatly impacts the control of arboviral diseases that are transmitted by vectors. The aim of the present study was to determine fauna of mosquitoes (Diptera: Culicidae) based on morphological and molecular (genomic) identification and their habitats in Lorestan province, Western Iran. Meanwhile mosquito samples were examined for arbovirus infection. Culicidae mosquitoes were caught in 2015 and 2016 from human homes, animal dwellings, storehouses and pit shelters in Lorestan province, Western Iran, using an oral aspirator (hand catch), total catch, human and animal bait and light trap methods. The samples were identified on the genus and species. Six species of Culex and eight species of Anopheles were caught. One complex species (Cx. pipiens complex) and a hybrid between Cx. pipiens pipiens biotype pipiens and Cx. pipiens pipiens biotype molestus were identified. Among all of the trapped mosquitoes (4211), 94.68% were from genus Culex mosquitoes (3987), which indicate that this genus is the dominant in Lorestan province, Western Iran. Anopheles comprised of 201 individuals out of the total catch. Arboviruses were not detected in these samples.

The risk of transmission of Crimean-Congo hemorrhagic fever virus from human cases to first-degree relatives.

To determine the infectivity of Crimean-Congo hemorrhagic fever (CCHF) virus via routine contacts between serologically confirmed cases and their close relatives from May 2005 up to March 2006, 79 serum samples of 57 close relatives of 12 newly diagnosed serologically confirmed CCHF cases in the Sistan-va-Baluchestan province of Iran were tested for IgG and IgM antibodies against CCHF virus using the enzyme-linked immunosorbent assay technique. Nine levels of contacts were considered: percutaneous contact with the patient's blood, cutaneous contact with the patient's blood, cutaneous contact with non-sanguineous body fluids, cutaneous contact with the patient's skin, sexual contact, eating at the same table, being a roommate of the patient, being a housemate of the patient, and living with the patient in the same building. Only one out of 57 relatives was positive for anti-CCHF IgG (1.8%, 95% confidence interval 0.0 to 9.8%). Thus, the infectivity of the virus via usual routine contacts with patients appears to be low.

Hyalomma anatolicum as the Main Infesting Tick in an Important Livestock Rearing Region, Central Area of Iran.

BACKGROUND: This study was carried out to determine the infestation of domestic ruminants to ticks in an important livestock-rearing region, located in central part of Iran. METHODS: Ticks were collected from cattle, sheep, and goats and then were identified with appropriate identification keys to species level in two different ecological regions of plains and mountain in 4 seasons in 2015. RESULTS: Totally 492 ticks from cattle, sheep, and goats in 34 herds were collected. Totally, 18.53% of domestic animals were infected by ticks. All ticks were belonged to family Ixodidae and classified into three genera and six species comprising Hyalomma anatolicum (38.83%), Hy. Asiaticum (23.37%), Hy. marginatum (2.85%), Hy. sp. (3.45%), Rhipicephalus sanguineus (14.02%) and Haemaphysalis sulcata (10.98%). Sex ratio of the collected specimens showed 241 (48.99%) male, 219 (44.51%) female and 32 (6.5%) nymph. CONCLUSION: Studied area is important for production of livestock and dairy products. Annually, many livestock products are exported to other parts from this region; therefore, it is very important to identify the infection rate of tick-borne diseases as well as safety factors on livestock.