Iranian Journal of Virology
Volume:3 Issue: 3, 2009

The ELISA titers in sera collected from animals were higher than those recorded by VNT. These results suggested that, in addition to neutralizing antibodies, the ELISA was measuring other classes of antibodies which did not neutralize FMDV in vitro. Also comparison of the VNT and B- ELISA results by t-test showed that the gamma irradiated inactivated FMDV antigen has unaltered antigenicity. The results of VNT and B-ELISA did not show significant differences (P>0.05). The sensitivity and specificity of B-ELISA in comparison to VNT for detection of anti FMDV type A 87/IRN antibody were calculated to be %94.73 and %90.9 respectively. FMDV type A87/IRN was propagated to a titer of 107.5/ml TCID50 and then irradiated by gamma irradiator. The irradiated 146 S antigen was purified by sucrose gradient centrifugation after inactivation, and used for inoculation of rabbits and guinea pigs. The anti-FMDV sera were collected from the animals. ELISA test was optimized and the titer of antibodies was compared with that of virus neutralization test (VNT) to determine the correlation between these techniques. The aim of this study was application of gamma irradiated FMDV antigen for liquid phase blocking sandwich ELISA (B-ELISA) to quantify guinea pig anti FMDV type A87/IRN antibody.

Influenza A virus subtype H9N2 have continued to circulate in domestic poultry farms in Asia since 1998. The virus circulates in live bird markets, missing link in epidemiology of avian influenza. Regarding previous studies on H9N2 viruses of Iran and having no data on this subject in Iranian live bird markets this study was conducted to analyze genetically hemagglutinin protein of H9N1 virus. A total of 500 tracheal and cloacal swabs from clinically healthy birds of Tehran's live bird markets were collected. Diagnostic RT-PCR was done on them using specific primers for subtype H9N2. Eight positive samples were selected for inoculation into 9-11 days SPF emberyonated eggs and the virus was grown and isolated. Amplification of the HA gene was carried out by PCR using two pairs of specific primers. PCR products were separated, purified and cloned. The products were sequenced and analyzed with M13 primers. They shared high amino acid homology with genes of other H9N2 viruses isolated previously in Asia and Iran. H9N2 viruses isolated from live bird markets were highly similar to viruses isolated from industrial poultry being circulated as early as 2001. The results suggest that a common source of H9N2 viruses is circulating in Iran.

NDV (Newcastle Disease Virus) is one of the viruses that cause disease in avian with severe economic losses in the poultry industry in many countries. Fusion protein (F) which plays a major role in the virus pathogenicity contains several regions that have a role in the fusion process. Mutation in the sequence of HR1 & HR2 regions of this protein prevents fusion of the virus to host cell. In addition, the proteins of HR1 and HR2 regions have antitumor properties that are related to their pathogenicity. In this investigation we used Newcastle disease virus NR43 isolate, from poultry diseases diagnostic department of Razi vaccine and serum research institute. RNA was extracted using SDS and proteinase K procedure. In the next step, RT-PCR was carried out and then cDNA cloned in pTZ57RT vector. After sequencing and alignmenting of the cDNA, a pair of proper primers for cloning HR1 in expression vector Pet32a(+) was designed. The HR1 expression was carried out by SDS–PAGE Western- Blotting in which the peptides were blotted onto nitrocellulose membrane using Ni-NTA anti His tag (1:1500 dilutions) coupled to HRP enzyme. A peptide with 23.76 kD/a molecular weight s peptide was obtained. By cloning and expression of HR1 region of protein F, it will be possible to express the whole gene that could be introduced as a novel vaccine against NDV.

Inhibition of viral growth in coinfected cells with two different viruses has been described. This phenomenon known as viral interference can occur in several virus host systems such as interference of enterovirus infection on poliovirus vaccine strains. In this study we superinfected reovirus infected HeLa cells with poliovirus to determine if poliovirus can replicate in such cells and form mature virus particles. Cells were infected first with reovirus then were reinfected with poliovirus. The amounts of viral particles were measured by electron microscopy and plaque assay titration. The amount of viral yield was also measured using the technique of real time RT-PCR for measuring the viral load in infected cells. In cells infected first with reovirus and then superinfected with polio virus, only reovirus particles were produced. Virus production was determined by assaying viral titer using the plaque assay technique and electron microscopy. There was no poliovirus particles observable in the superinfected cells. The amount of poliovirus load in reovirus infected cells was also drastically reduced. The growth of poliovirus was inhibited in reovirus infected cells and no infectious poliovirus particles could be observed. This observation could be important to consider in poliovirus vaccination program.

Since the produced recombinant proteins by molecular genetics techniques commonly have some limitations in the application, chimeric protein are introduced. Chimeric proteins have found widespread application for the study of protein folding, structure stability, function and immunogenisity. According to the known Immunomodulatory effect and structure of HSP70 molecule, full length human HSP70 was selected and a hybrid was made with HPV-E7gene.The recombinant plasmid pCDNA3.1/ E7-HSP70 was constructed using sequential PCR and cloning steps. The entire upstream and downstream sequences of the target molecules were synthesized separately. The sequential cloning was performed for cloning of the entire sequences of the target molecule of HPV16-E7 fragment in pcDNA3.1. Target DNA was visualized by staining with ethidium bromide. The Cos-7 cell lines were transfected with fusion proteins in 6 well microtiter plates. After 48 hours of transfection, the target cells were removed and to SDS-PAGE analysis for mRNA detection. Immunoreactivity of the protein product was assayed by Western blotting using monoclonal antibody. The sequencing analysis showed that E7 gene was fused in frame to the HSP70 gene in pcDNA3.1/E7-HSP70. Western blot analysis of recombinant fusion protein using HSP 70 monoclonal antibody showed desired band as expected. The chimeric structure was expressed in cells, as expected. The resulting E7- HSP70 fusion gene would be a useful construct for future research. In order to change and enhance of the tropism and immunogenicity of recombinant protein, chimeric E7- HSP70 Vector was constructed. Since monovalent molecule and vaccines were clinically ineffective or poorly immunogenic, so applications of covalently linked product are introduced. The successful expression of the E7- Hsp70 fusion protein can be used as a molecular target for establishment of DNA and recombinant protein vaccine in future research.