Alireza Sazmand

Designing and Modeling of Complex DNA Vaccine Based on Tropomyosin Protein of Boophilus Genus Tick

Boophilus tick is a bloodsucking ectoparasite that transfers some pathogens, reducing production and thus leading to economical losses in the cattle industry. Tropomyosin (TPM) protein is a salivary protein, has actin regulator activity, and plays an important role in immune reactions against parasites. In the current study, besides developing a safe, effective, and broad spectrum protective measure against Boophilus genus tick based on TPM protein, we attempted to minimize possible problems occurring in the design of polytopic vaccines. Briefly, the steps that were followed in the present study were as follows: retrieving sequences and finding the mutational/conservative regions, selecting consensus and high immunogenic epitopes of B and CD4+ T cells by different approaches, three-dimensional structure (3D structure) prediction and representation of epitopes and highly variable/conserve regions, designing vaccinal construct by fusion of B and T cell epitopes by special patterns and improving immunogenicity, evaluation of the constructs’ primary structure and posttranslational modification, calculation of hydrophobic regions, reverse translation, codon optimization, open reading frame checking, insertion of start/end codon, Kozak sequence, and finally constructing the DNA vaccine. Variation plot showed some shared epitopes among the ticks’ and mites’ species that some might be effective only in some species. Finally, by following the steps mentioned above, two constructs for B and T cells were achieved. Checking constructs revealed their reliability and efficacy for in vitro production and utilization. Successful in silico modeling is an essential step of designing vigorous vaccines. We developed a novel protective and therapeutic vaccine against Boophilus genus (based on TPM protein). At the next step, constructed DNA vaccine would be produced in vitro and administrated to cattle, and its potency to induction of immune response and protection against Boophilus genus as well as other ticks and mites will be evaluated.

GB Virus C/Hepatitis G Virus Envelope Glycoprotein E2: Computational Molecular Features and Immunoinformatics Study

Introduction: GB virus C (GBV-C) or hepatitis G virus (HGV) is an enveloped, RNA positive-stranded flavivirus-like particle. E2 envelope protein of GBV-C plays an important role in virus entry into the cytosol, genotyping and as a marker for diagnosing GBV-C infections. Also, there is discussion on relations between E2 protein and gp41 protein of HIV. The purposes of our study are to multi aspect molecular evaluation of GB virus C E2 protein from its characteristics, mutations, structures and antigenicity which would help to new directions for future researches. Evidence Acquisition: Briefly, steps followed here were; retrieving reference sequences of E2 protein, entropy plot evaluation for finding the mutational /conservative regions, analyzing potential Glycosylation, Phosphorylation and Palmitoylation sites, prediction of primary, secondary and tertiary structures, then amino acid distributions and transmembrane topology, prediction of T and B cell epitopes, and finally visualization of epitopes and variations regions in 3D structure. Results: Based on the entropy plot, 3 hypervariable regions (HVR) observed along E2 protein located in residues 133-135, 256-260 and 279-281. Analyzing primary structure of protein sequence revealed basic nature, instability, and low hydrophilicity of this protein. Transmembrane topology prediction showed that residues 257-270 presented outside, while residues 234- 256 and 271-293 were transmembrane regions. Just one N-glycosylation site, 5 potential phosphorylated peptides and two palmitoylation were found. Secondary structure revealed that this protein has 6 α-helix, 12 β-strand 17 Coil structures. Prediction of T-cell epitopes based on HLA-A*02:01 showed that epitope NH3-LLLDFVFVL-COOH is the best antigen icepitope. Comparative analysis for consensus B-cell epitopes regarding transmembrane topology, based on physico-chemical and machine learning approaches revealed that residue 231- 296 (NH2- EARLVPLILLLLWWWVNQLAVLGLPAVEAAVAGEVFAGPALSWCLGLPVVSMILGLANLVLYFRWL-COOH) is most effective and probable B cell epitope for E2 protein. Conclusions: The comprehensive analysis of a protein with important roles has never been easy, and in case of E2 envelope glycoprotein of HGV, there is no much data on its molecular and immunological features, clinical significance and its pathogenic potential in hepatitis or any other GBV-C related diseases. So, results of the present study may explain some structural, physiological and immunological functions of this protein in GBV-C, as well as designing new diagnostic kits and besides, help to better understandingE2 protein characteristic and other members of Flavivirus family, especially HCV.

Parasitic diseases of camels in Iran (1931–2017) – a literature review

Parasitic diseases of camels are major causes of impaired milk and meat production, decreases in performance or even death. Some camel parasites also represent a threat to human health. About 171,500 one-humped camels (Camelus dromedarius) and 100–300 two-humped camels (Camelus bactrianus) live in Iran. Knowledge of the biodiversity of their parasites is still limited. The present review covers all information about camel parasitic diseases in Iran published as dissertations and in both Iranian and international journals from 1931 to February 2017. Ten genera of Protozoa (Trypanosoma, Eimeria, Cryptosporidium, Toxoplasma, Neospora, Sarcocystis, Besnoitia, Theileria, Babesia and Balantidium), 48 helminth species detected in the digestive system, including three species of Trematoda, four species of Cestoda, and 41 species of Nematoda, as well as helminths from other organs – Echinococcus spp., Dictyocaulus filaria, Thelazia leesei, Dipetalonema evansi and Onchocerca fasciata – have so far been described in Iranian camels. Furthermore, 13 species of hard ticks, mange mites, the myiasis flies Cephalopina titillator and Wohlfahrtia magnifica, and immature stages of the Pentastomida Linguatula serrata have also been reported from camels of Iran. Camel parasitic diseases are a major issue in Iran in terms of economics and public health. The present review offers information for an integrated control programme against economically relevant parasites of camels.

Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus) annulatus larvae.

Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.

Morphological and molecular characterization of Onchocerca fasciata (Nematoda, Onchocercidae) from dromedary camels (Camelus dromedarius) in Iran

Skin nodules of Onchocerca fasciata Railliet and Henry, 1910 (Spirurida, Onchocercidae) are a common finding in dromedary camels, though with a minimal clinical impact. There is little information about the morphology, molecular make-up and pathological impact of this parasite. Onchocerca fasciata nodules (1.3–2.1 cm in diameter and 509–841 mg in weight) were detected on the neck region in 31.5% of dromedary camels examined in Kerman province, southeastern Iran. Of 38 isolated nodules, only 23 (60.5%) contained viable worms. Measurement and morphological analyses were performed on isolated female worms by light microscopy. The identification of O. fasciata specimens was confirmed by sequence analysis of two mitochondrial genes (12S rDNA and cox1), which showed 0.4% divergence from available O. fasciata sequences. In addition, a phylogeny of filarial nematodes was constructed, based on these two mitochondrial genes and five nuclear genes (18S rDNA, 28S rDNA, MyoHC, rbp1, hsp70); this indicated that O. fasciata belongs to clade ONC3 of Onchocercidae, with representatives of the genera Onchocerca and Dirofilaria. Within the genus Onchocerca, O. fasciata is grouped with bovine parasitic species and the human parasitic Onchocerca volvulus, which suggests an impact of domestication on the radiation of the genus. Data provided here on the distribution and morphology of O. fasciata contribute to the molecular identification and phylogenetic position of the species.