In the case of Newcastle disease virus, multiple factors such as host adaptation, immune response evasion, and selective pressures have been suggested to result in evolution of viruses and the emergence of genetic variants. Multiple studies on virus classification and global epidemiological links have yielded consistent data. Here, we have performed a molecular analysis study of circulating Newcastle disease viruses in Iran (1995-2016). According to evolutionary divergences, subgenotype VIg, VIj, VIIj, VIId, XIIIa and XIIId isolates have been circulating in the country during a 21-year period. Based on data analysis, VIg isolates shared highest sequence identity with Russian and Polish isolates of the VIg subgenotype, while VIj subgenotype isolates (2012) were most similar to a virus isolated in 2015 in India. Analysis of the evolutionary divergence of subgenotype VIIj suggests that Chinese and Ukrainian viruses may have played a crucial role in the emergence of VIIj isolates. Evolutionary difference studies also indicated that XIIIa isolates circulating in Iran may have caused the emergence of adapted variants of subgenotype XIIId. Therefore, we propose that the evolutionary and epidemiological study of virulent Newcastle disease viruses could help to provide accurate molecular data about variants circulating in the region, thus aiding in the design of more efficient recombinant vaccines.

Introduction: Leptospirosis, caused by infection with pathogenic Leptospira species, is one of the most prevalent zoonotic diseases in the world. Current leptospiral vaccines are mainly multivalent dead whole-cell mixtures made of several local dominant serovars. Therefore, design and construction of an efficient recombinant vaccine for leptospirosis control is very important. OmpL1 is an immunogenic porin protein that could be of special significance in vaccination and serodiagnosis for leptospirosis. Methodology: Three strains belonging to pathogenic L. interrogans were analyzed. The specific primers for proliferation of the ompL1 gene were designed. The amplified gene was cloned. In order to investigate the ompL1 nucleotide sequence and homological analysis of this gene, ompL1 genes cloned from standard vaccinal Leptospira serovars prevalent in Iran were sequenced and cloned. Results: PCR amplification of the ompL1 gene using the designed primers resulted in a 963 bp ompL1 gene product. The PCR based on the ompL1 gene detected all pathogenic reference serovars of Leptospira spp. tested. Based on alignment and phylogenetic analysis, although the ompL1 nucleotide sequence was slightly different within three vaccinal serovars (100%-85% identity), amino acid alignment of the OmpL1 proteins revealed that there would be inconsiderable difference among them. Conclusion: The ompL1 gene of the three isolates was well conserved, differing only by a total of 6 bp and the proteins by 2 amino acids. The cloned gene could be further used for expression and recombinant OmpL1 as an efficient and conserved antigen, and may be a useful vaccine candidate against leptospirosis in our region.

Background:Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis. Development of a new vaccine for tuberculosis requires immunogenic antigens capable of inducing suitable and long-lasting T cell immunity. The emergence of multidrugs and extensively drug-resistant strains of M. tuberculosis has made it a global public health concern.Objectives:DNA vaccine is a straightforward method to introduce antigens to the host. In the present study, two immunodominant mycobacterial antigens (Mtb32C and HBHA) were isolated and cloned into pcDNA3.1 (+) to design and construct a new DNA vaccine. This vector is capable of producing new potent chimeric protein.Materials and Methods:Mtb32C (Rv0125) and heparin-binding haemagglutinin adhesion (HBHA) genes were amplified using polymerase chain reaction (PCR) of M. tuberculosis H37Rv genome and ligated into pcDNA3.1 (+). Colony-PCR and restriction enzyme analysis were used to confirm the accuracy of the cloning procedure.Results:In the current study, recombinant pcDNA3.1 (+) vector containing Mtb32C and HBHA genes was successfully constructed. The desired size of DNA fragment was observed using single and double digestion methods.Conclusions:Mtb32C and HBHA genes were successfully isolated from H37Rv genome and cloned into an eukaryotic expression vector. This vector can be considered as a vaccine to evaluate immune responses in animal models.

Newcastle disease virus (NDV) is believed to be the cause of fatal poultry disease worldwide. The fusion (F) protein plays a key role in virus pathogenesis, and it is also used for Newcastle disease virus classification. In this study, we determined the complete coding sequence of the F gene in new velogenic NDV isolates with an intravenous pathogenicity index (IVPI) of 1.8 and a mean death time (MDT) of 72 or 48 h. Complete sequences of the F genes of new Iranian isolates were amplified and sequenced in both directions. These isolates were compared to 195 nucleotide sequences from GenBank (available as of 07/17/2016). A phylogenetic tree was constructed for the F gene, using MEGA6 software with statistical analysis based on 500 bootstrap replicates. Evolutionary distances revealed that the new virulent isolates from Iran belonged to genotype VII in a new distinct sub-genotype named VII-(j). This new sub-genotype showed 3% divergence from genotype VIId. Recombination analysis showed that these new isolates were not recombinant NDVs.

BackgroundLeptospirosis is a worldwide zoonosis caused by pathogenic Leptospira species. A major challenge of this disease is the application of basic research to improve diagnostic methods and related vaccine development. Outer membrane proteins of Leptospira are potential candidates that may be useful as diagnostic or immunogenic factors in treatment and analysis of the disease.ObjectivesTo develop an effective subunit vaccine against prevalent pathogenic Leptospira species, we sequenced and analyzed the LipL32 gene from three different Leptospira interrogans (L.interrogans) vaccinal serovars in Iran.Materials and MethodsFollowing DNA extraction from these three serovars, the related LipL32 genes were amplified and cloned in the pTZ57R/T vector. Recombinant clones were confirmed by colony- PCR and DNA sequencing. The related sequences were subjected to homology analysis by comparing them to sequences in the Genbank database.ResultsThe LipL32 sequences were >94% homologous among the vaccinal and other pathogenic Leptospira serovars in GenBank. This result indicates the conservation of this gene within the pathogenic Leptospires.ConclusionsThe cloned gene in this study may provide a potentially suitable platform for development of a variety of applications such as serological diagnostic tests or recombinant vaccines against leptospirosis.

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

ObjectivesBrucellosis is a major zoonotic disease that poses a significant public health threat worldwide. The classical bacteriological detection process used to identify Brucella spp. is difficult and time-consuming. This study aimed to develop a novel molecular assay for detecting brucellosis.MethodsAll complete sequences of chromosome 1 with 2.1-Mbp lengths were compared among all available Brucella sequences. A unique repeat sequence (URS) locus on chromosome 1 could differentiate Brucella abortus from Brucella melitensis. A primer set was designed to flank the unique locus. A total of 136 lymph nodes and blood samples were evaluated and classified by the URS-polymerase chain reaction (PCR) method in 2013–2014.ResultsBiochemical tests and bacteriophage typing as the golden standard indicated that all Brucella spp. isolates were B. melitensis biovar 1 and B. abortus biovar 3. The PCR results were the same as the bacteriological method for detecting Brucella spp. The sensitivity and specificity of the URS-PCR method make it suitable for detecting B. abortus and B. melitensis.ConclusionQuick detection of B. abortus and B. melitensis can provide the most effective strategies for control of these bacteria. The advantage of this method over other presented methods is that both B. abortus and B. melitensis are detectable in a single test tube. Furthermore, this method covered 100% of all B. melitensis and B. abortus biotypes. The development of this URS-PCR method is the first step toward the development of a novel kit for the molecular identification of B. abortus and B. melitensis.

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