Pneumoviruses are responsible for significant respiratory disease in their hosts and represent a major problem for human and animal health. Pneumoviruses are members of the family Paramyxoviridae، subfamily Pneumovirinae and the virus particles consist of a negative-sense، nonsegmented RNA genome within a helical nucleocapsid structure enveloped in a lipid membrane derived from the host cell. Over the past four decades much work has extended our understanding of the molecular biology and pathogenesis of pneumoviruses but despite this only limited treatments and prophylaxis are available. The human pathogen، respiratory syncytial virus (hRSV) which belongs to the genus of Pneumovirus is the best characterized of the subfamily. HRSV is the major cause of hospitalisation of very young children with respiratory disease worldwide. No vaccine is available though new treatments offer some respite for children in the highest risk groups، the immunocompromised and children with congenital heart disease. The recently discovered human pathogen human metapneumovirus (hMPV) belongs to the genus Metapneumovirus and recent data indicates that this virus is second only to hRSV in terms of disease impact. The pneumoviruses also include agents of veterinary importance such as bovine respiratory syncytial virus (bRSV)، ovine and caprine RSV، and pneumonia virus of mice (PVM: all in the genus Pneumovirus) and avian metapneumovirus (APV: genus Metapneumovirus). The development of reverse genetics systems for negative strand RNA viruses has opened the possibility of manipulating the virus genomes to identify genes involved in pathogenesis and to explore the biological consequences of specific mutations. This information is informing the rational design of new vaccines. These plasmid-based systems have shown that for all paramyxoviruses the N، P and L proteins are necessary and sufficient for RNA replication. However، the pneumoviruses differ from the other family members in that fully efficient transcription from the virus genome requires the presence of an additional protein encoded by the M2 gene. The present article reviews pneumovirus biology and molecular genetics including a discussion of current concepts of Pneumovirus reverse genetics.

In the recent decade, the reverse genetics method has been broadly used for rescue of negative-stranded RNA viruses from cDNA or viral minigenomes. This technique has been applied to study different steps in virus replication and virus-host interactions. Reverse genetics could also be implemented for design of new vaccines. The T7 RNA polymerase activity as well as virus (nucleocapsid protein) N, (phosphoprotein) P and (Large) L proteins are necessary to rescue the virus or viral minigenome. Measles virus is a negative-stranded non-segmented RNA virus. There are useful vaccine strains to prevent measles disease.. Here, we describe the construction of a new helper cell line for rescue of measles virus minigenome. The helper cell line stably expresses T7 RNA polymerase as well as measles virus N and P proteins by tricistronic mRNA.. For rescue of measles virus minigenome a stable helper cell line by using tricistronic expression vector was developed which expressed T7 RNA polymerase as well as measles virus N and P proteins. To construct the tricistronic expression vector, T7 RNA polymerase gene was cloned after cytomegalovirus (CMV) promoter and measles virus N and P proteins were under control of IRES (internal ribosome entry site) sequences.. Our results indicated that measles virus minigenome could be rescued in this constructed helper cell line.. Through this system, the measles virus minigenome was rescued. Further studies are necessary to improve the rescue efficiency. This may be possible by replacing the CMV promoter with the T7 promoter..

The genes controlling meiotic progression in plants and not affecting mitotic progression are most widely studied in maize Zea mays and cruciferous plant Arabidopsis thaliana. These include the genes controlling the differentiation of somatic cells into sporogenous ones and meiosis-initiating genes, genes encoding meiosis-specific proteins of chromosomes and synaptonemal complexes, genes of mediator proteins and enzymes of meiotic DNA recombination and crossover, and genes controlling meiosis-specific behavior of centromeres and the course of two meiotic divisions. A large number of such genes have been cloned and studied at the molecular level. The studies of meiotic genes in rice Oriza sativa are actively developing, while studies of corresponding genes in barley Hordeum vulgare, rye Secale cereale, tomato Solanum lycopersicum, and hexaploid wheat Triticum aestivum are less advanced. To identify meiotic genes, chemical and insertional mutagenesis, genetic and cytological analysis, genomic and proteomic studies, methods of reverse genetics, and bioinformatics are used.

Rabies is a zoonotic disease, endemic mostly in Asia and Africa. Rabies virus belongs to the family Rhabdoviridae, genus Lyssavirus. Because infection with rabies virus has no cure and is life-threatening, vaccination is an important preventive measure to combat this disease in humans and animals. On the other hand, considering the limited availability of rabies vaccines in some of less developed countries with a higher rate of human rabies death each year, the need for an alternative strategy to produce a cost-effective and immunogenic vaccine against this disease is essential. In this review, we provide a brief overview of rabies vaccine development and its recent basic research. We also describe how viral vectors such as poxvirus vector, adenovirus replicons, and reverse genetics are manipulated for efficient novel formulated vaccines against this infection and we highlight possible future developments</span></span></span></span>

Human rotavirus (RV) is responsible for most cases of acute gastroenteritis in infants, worldwide. Today, in vitro transcription (IVT) assay is widely used to develop efficient RNA for the biological experiments such as gene function analysis and reverse genetics. The aim of this study was to develop optimal full-length transcripts of the VP7 segment, using in vitro transcription assay.
Special primers were designed in order to synthesize VP7 sequence of sense RNA in the process of IVT using T7 RNA polymerase. RT-PCR was performed using forward and reverse primers, containing T7 promoter sequence and BstUI restriction enzyme site, respectively. In order to synthesize ssRNA VP7, in accordance with the IVT technique, RV4-VP7 fragment was subcloned into PTZ57 R/T plasmid and digested by BstUI enzyme.
The sequencing of the VP7 gene showed 99% identity withVP7 gene of rotavirus RV4 strain (Sequence ID: M64666.1). The analysis of purity of DNA fragment and ssRNA VP7 segment revealed that OD ratio of A260/A280 and quantity of nucleic acids were (1.9, 0.036 µg/µL) and (2.02, 0.98 µg/µL), respectively.
In the present study, a modified methodology of RNA synthetase was described by IVT assay, using T7RNA polymerase in order to transcribe the full-length transcripts of human VP7-RV4 strain. This method is applicable for reverse genetic approaches, especially for the production of reassortant RV vaccine.

Rabies is a deadly zoonotic disease that is caused by the rabies virus. The virus can infect and disrupt the central nervous system of a rabid patient. The rabies virus is a neurotropic single stranded RNA virus. Glycoprotein (G) is the most important protein that binds to the cellular receptors and also induces an immune response against the virus in the host. Using reverse genetics technology, the glycoprotein gene could be modified and a virus with higher immunogenicity or lower pathogenicity.

In this study, we designed a mutation in the sequence of glycoprotein gene using a software, on the main antigenic site II of the Pasteur virus strain at the position of 42-34 amino acids. Agene fragment in the cloning vector containing the rabies virus genome was replaced by the synthesized construct containing the altered gene by two restricted enzymes, and then cloned. The T7-BHK cell under the T7 phage promoter control was transfected to express the glycoprotein gene, along with the construct and vectors expressing the N, P, and L genes of the rabies virus as well as the full genome. After expressing and confirming viral genes, it was cultured and amplified in BSR cell.

after cloning and expression of the recombinant virus in the target cell, the vector containing the mutated gene led to the rescue of the recombinant virus. The recombinant virus cultured and propagated in the BSR cells, then the genome was extracted and finally confirmed by sequencing.

The rescued recombinant virus can be used for research studies or in the vaccines manufacturing, provide that the antigenicity is maintained or increased.

Recently, the use of T7 RNA polymerase instead of other viral and cellular promoters is increasing due to high efficacy of transcription in the cell cytoplasm by this polymerase. In order to translate the transcripts produced by T7 RNA polymerase in mammalian cell lines, it is necessary to include Internal Ribosome Entry Site (IRES) sequences. In addition, if sequence of poly A signal would be included after interested gene, the rate of expression could be increased in the cells. For expression of eGFP in HEK-293 and T7-BHK cells by T7 RNA polymerase, the sequence of eGFP as well as IRES sequences upstream of eGFP gene and poly A signal were inserted into a pUC57 plasmid. On the other hand, gene of T7 RNA polymerase was cloned into modified pIRES2-EGFP plasmid. Then, the constructed plasmids were transfected into HEK-293 cells. T7-BHK cell was used for control of T7 RNA polymerase activity. Our results showed that using T7 RNA polymerase for expression of foreign genes in mammalian cell lines is highly efficient. Highly efficient eGFP expression in HEK-293 cells showed that T7 RNA polymerase could be used for cytoplasmic RNA transcription such as production of anti-cancer proteins and oncolytic viral genomic RNA by reverse genetics.

Strictosidine synthase-like (SSL) is a group of gene family in the Arabidopsis thaliana genome, which their orthologous in other plants, such as Catharanthus roseus, are key enzymes in the monoterpenoid indole alkaloid biosynthesis pathway. Arabidopsis SSL6 gene, a member of SSL family, has been induced significantly by various stresses and signaling molecules. In this study, the reverse genetics approaches (T-DNA insertion) are used to determine the function of the SSL6 gene in response to the salt stress. The experiment was conducted using a factorial experiment based on the completely randomized design with two factors and three replications. These factors were included two genotypes (Col-0 and ssl6) and three NaCl treatments (0 mM, 100mM and 200 mM). Compared with the wild-type, the ssl6 mutant shows significantly increased proline accumulation, anthocyanin content and reduced malondialdehyde (MDA) content in response to the salt stress. Real-Time PCR analysis revealed that the expression levels of SOS genes were upregulated significantly in ssl7 compared with the expression in Col-0 in response to 150 mM NaCl at 3 h and 6 h after the treatment. Totally, these results suggest that SSL6 might have a negative regulatory role in response to salt stress in Arabidopsis.

Up to now, several attenuated measles virus vaccine strains derived from the Edmonston B vaccine consisting of Schwarz/Moraten, Zagreb, and AIK-C have been introduced for the rescue of their relative viruses through reverse genetics. In most studies, the measles virus rescue was done by supplying a cell line that expresses T7 RNA polymerase and measles virus N and P proteins as accessory proteins. The present study aimed to evaluate the rescue efficiency of the recombinant measles virus AIK-C vaccine strain by using a tricistronic expression plasmid. In this study, the rescue of the recombinant measles virus AIK-C vaccine strain was performed by co-transfection of three plasmids, including the cloned antigenomic cDNA of measles virus, a tricistronic expression plasmid that contained T7 RNA polymerase and measles virus N and P genes, and measles virus L polymerase expression plasmid. To increase the rescue efficiency, the transfected HEK-293 cells were co-cultured with Vero cells. As a result, the use of tricistronic expression plasmid that concomitantly encoded three necessary genes for the rescue of the measles virus led to the viral cytopathic effect with high efficacy five days post-transfection. Finally, the co-culture of transfected HEK-293 cells with Vero cells showed a relatively fast induction of viral cytopathic effect.