Hantavirus infection in Iranian patients suspected to viral hemorrhagic fever.

BACKGROUND: Hantaviruses are a group of emerging pathogens causing hemorrhagic fever with renal syndrome and Hantavirus cardiopulmonary syndrome in human. This study was conducted to investigate Hantavirus infection among Iranian viral hemorrhagic fever suspected patients. METHODS: From April 2014 to June 2016, 113 cases from 25 different provinces of Iran were analyzed for Hantavirus infection by IgM/IgG ELISA and pan-Hantavirus RT-PCR tests. RESULTS: Although, viral genome was detected in none of the subjects, IgM and IgG antibodies were detected in 19 and 4 cases, respectively. Differentiation of the anti-Hantavirus antibodies according to virus species by EUROLINE Anti-Hantavirus Profile Kit revealed three Puumala virus IgM positive, one Hantaan virus IgM positive, one Hantaan virus IgM borderline, and two Puumala virus IgG borderline cases. CONCLUSIONS: This study demonstrates the circulation of Hantaviruses in Iran and calls for further investigations of these life-threatening viruses in the country.

Imported cases of Chikungunya virus in Iran.

BACKGROUND: Chikungunya virus (CHIKV) is a widespread mosquito-borne virus representing a serious challenge to public health. The largest outbreak in the Middle-East was recorded in 2016-2017 in Pakistan. Sistan and Baluchistan Province of Iran shares a wide border with Pakistan; accordingly, introduction of CHIKV from Pakistan to Iran seems to be probable. The current study is aimed at investigating CHIKV infection in Sistan and Baluchistan Province. METHODS: Between April 2017 and June 2018, a total of 159 serum samples of CHIK suspected cases from 10 cities of Sistan and Baluchistan Province were tested by molecular and serological assays. Samples obtained up to 4 days after onset of illness were tested by real time PCR (n = 8). Samples collected 5-10 days after disease onset were subjected to ELISA, as well as real time PCR tests (n = 72). Samples obtained after the 10th day of disease onset were tested by only ELISA (n = 79). Phylogenetic analysis of real time PCR positive samples was carried out by sequencing of a 1014-bp region of Envelope 1 gene (E1 gene). Chi-square and independent t tests were used to evaluate the association between variables and CHIKV infection. RESULTS: In total, 40 (25.1%) out of 159 samples tested positive either by real time PCR or ELISA tests.Out of 151 samples serologically analyzed, 19 (12.6%) and 28 (18.6%) cases were positive for anti-CHIKV IgM and anti-CHIKV IgG antibodies, respectively. Of 80 samples tested by real time PCR, CHIKV RNA was detected in 11 (13.7%) sera, all of them had recent travel history to Pakistan. Additionally, phylogenetic analysis of 5 samples indicated their similarity with recent isolates of Pakistan outbreak 2016-2017 belonging to Indian Ocean sub-lineage of ECSA genotype. A significant correlation between abroad travel history and CHIKV infection was observed (P < 0.001). The most common clinical symptoms included fever, arthralgia/arthritis, myalgia, headache, and chill. CONCLUSIONS: These results present substantial evidence of CHIKV introduction to Iran from Pakistan and emphasize the need for the enhancement of surveillance system and preventive measures.

Seroprevalence of West Nile virus in Khuzestan province, southwestern Iran, 2016-2017.

BACKGROUND & OBJECTIVES: West Nile virus (WNV) is a neurotropic Flavivirus transmitted to humans through mosquito bites. As there is no specific antiviral treatment or approved vaccine against WNV, control and prevention of the infection is the best strategy to reduce the burden of WNV-related diseases. The circulation of WNV has been indicated in several regions of Iran including the Khuzestan province. Considering the complex ecology of WNV, the latest data are necessary for the implementation of preventive measures. Therefore, the present study was designed to provide updated information on the seroepidemiology of WNV in Khuzestan province. METHODS: A total of 408 sera were taken from volunteers living in Khuzestan. The presence of specific immunoglobulin G (IgG) antibody against WNV was tested by the enzyme-linked immunosorbent assay (ELISA) method. All the data and participants' demographic information were analyzed by SPSS and Esri's ArcMap GIS software programs. RESULTS: Anti-WNV IgG antibody was detected in 97 (23.8%) out of the 408 tested sera. The highest seropositivity rate was observed in cases aged between 20-29 yr and the lowest seropositivity rate was seen in those <19 yr of age (p = 0.001). There was no statistically significant association between WNV infection and gender, occupation, and educational level. The majority of positive cases were from the city of Ahvaz (47 cases, 48.4%) and Andimeshk (32 cases, 33%). INTERPRETATION & CONCLUSION: This study supports the earlier findings suggesting the circulation of WNV in Khuzestan province. Therefore, the implementation of an integrated surveillance system and training of health care workers and general population regarding the infection would be valuable to reduce the burden of possible outbreaks.

Crimean-Congo hemorrhagic fever among children in Iran.

Crimean-Congo hemorrhagic fever (CCHF) is a viral zoonotic disease which is endemic in Iran. The etiological agent of CCHF is an RNA virus belonging to the genus Nairovirus of the family Bunyaviridae. CCHF virus (CCHFV) can be transmitted to humans through bites from infected ticks and direct contact with infected blood or tissues. Although the disease has been observed in different age groups, the rate of disease is lower in children and elderly. This study was designed to characterize CCHFV-infected children in Iran. Between 2000 and 2016, a total of 908 CCHF suspected cases (in children less than 19 years old) were evaluated for CCHFV infection by CCHF IgM ELISA and RT-PCR. CCHFV infection was observed in 161 (17.73%) of subjects. Most CCHF positive children were male (70.8%) and >15 years of age (65.8%). Contact with livestock was the main risk factor (35.4%). Sistan and Baluchestan provinces had the highest frequency within the infected cohort (68.3%). The overall mortality rate was 11.8%. This study also revealed a significant reduction in CCHF-fatality rates in Iranian children when compared to earlier studies in Iran. Having contact with livestock was the major risk factor and CCHF was more common in male children of an older age.

Evaluation of first rapid diagnostic kit for Anti-Crimean-Congo Hemorrhagic Fever virus IgM antibody using clinical samples from Iran.

Crimean-Congo Hemorrhagic Fever (CCHF) is a potentially fatal tick-borne viral disease, which is in the WHO list for emerging infections likely to cause major epidemics in the near future. Early diagnosis of CCHF is very important for both patient treatment and infection control. An efficient CCHF rapid test therefore is of great significance. This study was conducted to evaluate the sensitivity and specificity of the first CCHF rapid diagnostic kit (CCHF Sero K-SeT, CORIS BioConcept, Belgium) for detection of IgM specific antibody in patients' sera or plasma, using 87 clinical serum samples from Iranian patients. Although the assay showed an acceptable specificity of 92.9% (13/14), a low sensitivity rate of 39.7% (29/73) was observed. There was no association between the results of CCHF rapid diagnostic kit and the genotype of CCHF virus. This evaluation revealed that the CCHF Sero K-SeT is not suitable for screening of CCHF suspected cases due to its poor sensitivity.

Epidemiology of West Nile Virus in the Eastern Mediterranean region: A systematic review.

BACKGROUND: West Nile Virus (WNV), a member of the genus Flavivirus, is one of the most widely distributed arboviruses in the world. Despite some evidence for circulation of WNV in countries summarized by the World Health Organization as the Eastern Mediterrian Regional Office (EMRO), comprehensive knowledge about its epidemiology remains largely unknown. This study aims to provide a concise review of the published literature on WNV infections in the Eastern Mediterranean Regional Office of WHO (EMRO). METHODOLOGY/PRINCIPAL FINDINGS: A systematic review of WNV prevalence studies on humans, animals and vectors in the EMRO region was performed by searching: Web of Science, Science Direct, Scopus, PubMed, Embase and Google Scholar. Finally, 77 citations were included, comprising 35 seroprevalence studies on general population (24460 individuals), 15 prevalence studies among patients (3439 individuals), 22 seroprevalence studies among animals (10309 animals), and 9 studies on vectors (184242 vector species). Of the 22 countries in this region, five had no data on WNV infection among different populations. These countries include Kuwait, Bahrain, Oman, Syria and Somalia. On the other hand, among countries with available data, WNV-specific antibodies were detected in the general population of all investigated countries including Djibouti (0.3-60%), Egypt (1-61%), Iran (0-30%), Iraq (11.6-15.1%), Jordan (8%), Lebanon (0.5-1%), Libya (2.3%), Morocco (0-18.8%), Pakistan (0.6-65.0%), Sudan (2.2-47%), and Tunisia (4.3-31.1%). WNV RNA were also detected in patient populations of Iran (1.2%), Pakistan (33.3%), and Tunisia (5.3% -15.9%). WNV-specific antibodies were also detected in a wide range of animal species. The highest seropositivity rate was observed among equids (100% in Morocco) and dogs (96% in Morocco). The highest seroprevalence among birds was seen in Tunisia (23%). In addition, WNV infection was detected in mosquitoes (Culex, and Aedes) and ticks (Argas reflexus hermanni). The primary vector of WNV (Culex pipiens s.l.) was detected in Djibouti, Egypt, Iran and Tunisia, and in mosquitoes of all these countries, WNV was demonstrated. CONCLUSIONS: This first systematic regional assessment of WNV prevalence provides evidence to support the circulation of WNV in the EMRO region as nearly all studies showed evidence of WNV infection in human as well as animal/vector populations. These findings highlight the need for continued prevention and control strategies and the collection of epidemiologic data for WNV epidemic status, especially in countries that lack reliable surveillance systems.

Production of human epidermal growth factor using adenoviral based system.

Epidermal growth factor (EGF), a growth factor involved in cell growth and differentiation, is a small polypeptide with molecular weight of approximately 6 kDa known to be present in a number of different mammalian species. Experimental studies in animals and humans have demonstrated that the topical application of EGF accelerates the rate of epidermal regeneration of partial-thickness wounds and second-degree burns. Due to its commercial applications, Human EGF (hEGF) has been cloned in several forms. In the present study, adenoviral based expression system was used to produce biologically active recombinant hEGF. The presence of secreted recombinant hEGF was confirmed by a dot blot and its expression level was determined by enzyme-linked immuno-sorbent assay. Moreover, biological activity of secreted hEGF was evaluated by a proliferation assay performed on A549 cells. For production of hEGF in a secretory form, a chimeric gene coding for the hEGF fused to the signal peptide was expressed using adenoviral based method. This method enables the production of hEGF at the site of interest and moreover it could be used for cell proliferation and differentiation assays in tissue engineering research experiments instead of using commercially available EGF.

Expression of a biotin acceptor peptide-containing protein with potential incorporation on the lentiviral envelope as a viral surface engineering platform.

Lentiviral vectors are among the promising viral based-vectors in gene therapy applications, but the efficiency of their targeting needs to be improved. (Strept)avidin-biotin adaptor system is a novel approach to modify the lentiviral envelope for better targeting properties. Herein, we describe utilization of this adaptor system by designing a candidate envelope protein-bearing biotin acceptor peptide (BAP) and evaluation of its expression in 293T cells. To this end, a DNA sequence containing flexible linkers, a 15-aminoacids BAP and specific membrane regions of a viral protein was designed and synthesized in tandem. The synthesized gene was amplified with polymerase chain reaction to include BglII and SalI restriction sites and subcloned into the same sites of pDisplay vector in frame with HA-tag and myc epitope to construct the pDis-GS-BAP. 293T cells were transfected with pDis-GS-BAP and expression of resulting protein (dis-GS-BAP) was evaluated by Western blotting using anti-HA tag antibody. Efficiency of transfection procedure was evaluated by pEGFP-N1 vector and tracking for green fluorescent protein expression via fluorescence microscopy. Restriction analysis and DNA sequencing confirmed the precision of cloning steps. Fluorescence microscopy indicated above 70% transfection efficiency and Western blot analysis of pDis-GS-BAP-transfected 293T cells showed a protein band of approximately 17 kDa corresponding to the predicted size of dis-GS-BAP protein. These promising results indicate the possibility of cell surface expression and further biotinylation of dis-GS-BAP protein in ongoing studies.

A novel method to produce Influenza A virus matrix protein M1 Capsid Like Particles (CLPs).

Avian influenza viruses represent a growing threat for an influenza pandemic. The currently licensed influenza vaccines have inherent drawbacks which has led many research groups to explore different approaches of vaccine development among which Virus Like particles (VLPs) seem like a promising alternative in the near future. Although it is known that the Matrix 1 protein (M1) of influenza plays an essential role in VLP formation and it is documented that M1 is able to form dimers, it is not clear if M1 is capable of forming higher order structures without the interference of other influenza proteins or cell derived envelope. Here, for the first time we have demonstrated that expression of M1 alone is enough to form a Capsid Like Particle (CLP) without the requirement of any other external factor.