h. keyvanfar

Canine parvovirus infection is the most highly infectious in dogs younger than six months. Our study aimed to design and optimize an In-house PCR Assay for Rapid Detection of parvovirus type 2 and compares it with REAL-TIME PCR and LAMP Assay and phylogenetic analysis. The virulence gene selected for the categories was vp2 for CPV-2. PCR products were cloned in pTZ57R/T plasmid for preparation of positive control. Determination of the specificity of primers was done with the negative control virus genomes, and the limit of detection was determined for the Homemade PCR, REAL-TIME PCR, LAMP, and to perform a phylogenetic study using partial vp2 gene sequences. Added analysis of PCR products using agarose gel electrophoresis for the vp2 gene showed 485bp, and GAPDH 900 bp bands, respective amplification using negative control genomes as template was negative. The least detectable copy number for the vp2 gene in a 25 µl PCR reaction equals 19 copies by homemade PCR, LAMP, and REAL-TIME PCR 25 and 21 copies, respectively. The phylogenetic analysis for the five field sequences formed three distinct clusters. The in-house PCR has advantages such as high specificity, sensitivity, and the ability to detect major CPV-2 pathogens. This assay may replace the previous laboratory methods and work as an essential supplement to the more time-consuming assays. Phylogenetic analysis is necessary for epidemiological studies to control and prevent disease.

Peste des Petits Ruminunts (PPR) is an acute and contagious viral disease with rapid infection and transmission in sheep and goats. Vaccination of livestock is the best solution to prevent the spread and control of the disease. The aim of this study was to evaluate the immune response derived from vaccination with six formulations of inactivated PPR vaccine in goats and sheep using a completely randomized design with 2 × 2 factorial arrangement. The attenuated PPR virus was amplified in a highly manipulated suspension lymphoid cell having a high passage potential, and then inactivated using two inactivators (4 mM bromoethylene imine [BEI] or 3% hydrogen peroxide [H2O2]). Thereafter each inactivated virus was formulated using three different helpers (aluminum hydroxide, aluminum phosphate, and a 50% mixture of each of these two adjuvants). Iimmunogenicity and safety of the formulated candidate vaccines were evaluated by respective two doses of 0.1 and 1 (10-fold doses) ml of the vaccines administrated into 38 sheep and 28 goats. Each dose was subcutaneously administrated on 0 and 21 days of the experiment and post vaccination sera antibody titer against PPR virus was measured on days 0, 14, 21, 35, 42 and 60. The results showed that the mean antibody titer against PPR virus was not influenced neither by inactivator nor adjuvant up to 21- and 60-days post vaccination (P> 0.05). Also, no significant interaction was observed between different inactivators and adjuvants (P> 0.05). The appropriate antibody titers observed in different experimental groups indicated a good immunization of the formulated vaccines; however, their efficacy remained to be investigated using challenge test. Also, due to the lack of carcinogenicity, cheapness and environmental compatibility reported for H2O2, it is nominated as an inactivator in production inactivated PPR vaccine.

Lumpy skin disease (LSD) is a highly contagious disease of cattle and buffaloes that cause considerable economic losses to the livestock industry in endemic regions. Although the virus neutralization index (VNI) is the gold standard test for the detection of LSD antibodies, the use of this test in seroepidemiology studies and serosurveillance is laborious and time-consuming. Accordingly, for serodiagnosis of capripox diseases, ELISA assay has been developed, but the lack of information about its performance and efficiency is one of the limiting factors in the use of this test in seroepidemiological studies. In this study, the diagnosis agreement between a recently developed ELISA based on P32 recombinant protein and the VNI method, as a gold standard for serological diagnosis of Capripox virus, was evaluated using 95 sera samples including 25 negative control, 14 positive control, and 56 suspected cattle sera. Result showed that all the negative control sera assessed by VNI and ELISA had no antibody titers against LSD. All the 14 (100%) positive control sera had VNI values ≥1.5 and were considered as the positive sera; however, by ELISA method, 13 samples (93%) were diagnosed as the positive. Among the 56 samples collected from LSD-suspected cattle, 16 samples (29%) were detected as the positive by VNI, whereas 13 samples (23%) were detected as the positive sera by ELISA method. There was no statistically significant difference between the percentages of negative or positive samples assessed by ELISA or VNI method (P>0.05), associated with the Kappa index value of 0.71 showing the substantial agreement. These findings indicating an acceptable agreement and performance of the developed ELISA system based on P32 recombinant protein in comparison to VNI method for diagnosing antibody against LSD virus in cattle.

Peste des petits ruminants (PPR) is a highly contagious disease that is considered a major threat to the small livestock industry. Although vaccination via live-attenuated PPR vaccine is a main controlling strategy in the endemic area, during PPR eradication process, the inactivated PPR vaccine (iPPRV) is recommended. This study aimed to compare the inactivation kinetics of the PPR virus via different inactivants and immunogenicity evaluations of the iPPRV formulated vaccine in mice. The vaccinal live PPR virus was inactivated by either H2O2 or binary ethylenimine (BEI (at two concentrations of 1 or 4 mM. Thereafter, the inactivated virus was formulated with different adjuvants, including aluminum hydroxide (AH), aluminum phosphate (AP), and a mixture of AH and AP that were intraperitoneally (IP) administrated (0.1 mL) to 90 BALB/c mice in a completely randomized design and 3×3 factorial arrangement (9 animals per group). The booster vaccination was carried out in all animals 21 days after the primary vaccination. Results showed that the PPR virus was successfully inactivated by all the inactivation agents; however, the time of complete virus inactivation was estimated to be 482, 295, and 495 min post-treatment initiation for 1 mM BEI, 4 mM BEI, and H2O2, respectively. The main effect of inactivant on antibody titers against PPR virus that was measured after 42days post-immunization in mice was significant (P<0.05); however, the adjuvant and interaction effect of inactivator×adjuvant were not effective(P>0.05). Inactivation by 1 mM BEI was associated with a higher antibody titer against PPR virus (P<0.05) in comparison with both 4 mM BEI and H2O2 (2.51 vs. 2.25 and 2.22, respectively). Meanwhile, there were no significant differences among the used adjuvants in terms of eliciting antibody response against PPR virus. In conclusion, the use of 1 mM BEI in combination of AH, AP, or a mixture of AH and AP was associated with a higher immune response against PPR virus in mice. However, the appropriate inactivation kinetic of the virus and immunogenicity associated with the use of H2O2, as well as its biocompatibility property and better cost-benefit, nominated H2O2 to be used in iPPR preparation; however, more investigations are required in target animals.

As notifiable diseases, lumpy skin disease (LSD), sheep pox (SPP), and goat pox (GTP) are associated with a profound effect on cattle, sheep, and goat farming industries. Development of the ELISA method could effectively facilitate serodiagnosis of the infected animals. This study aimed to develop an ELISA system based on the recombinant full-length and truncated P32 protein (Tr.P32) of goat pox virus. The P32 protein was expressed in Rosetta strain of E. coli using pET24a+ vector and evaluated by SDS-PAGE and Western blotting. Then, Tr.P32 was purified by Ni-NTA affinity chromatography under denaturing conditions and used to develop a capripoxvirus-specific ELISA. Checkerboard titration and receiver-operating characteristic (ROC) analysis were used to optimize the ELISA system and determine diagnostic specificity and sensitivity, respectively. The diagnostic potential of the developed ELISA was evaluated using positive and negative control sera collected from goat, sheep, and cattle. Results showed that the expression level of full-length P32 recombinant protein was negligible, while Tr.P32, a ~ 31 kDa recombinant protein, was expressed up to 0.270-0.300 mg/200 mL of culture media. The results of checkerboard titration revealed that 675 ng/well of Tr.P32 antigen and 1:10 dilution of control sera (anti GTPV HIS and healthy goat sera) caused maximum difference in absorbance between positive and negative goat sera. The recombinant Tr.P32 showed good reactions with antibodies against GTP virus (GTPV), SPP virus (SPPV), and LSD virus (LSDV), whereas no cross-reactions with anti-Orf virus antibodies were detected. By comparing with the neutralization index (NI), cut off, diagnostic sensitivity and specificity of the developed indirect-ELISA were estimated, 0.397, 94% and 96.6%, respectively. These findings indicate that the ELISA system based on Tr.P32 protein could potentially be used in sero-surveillance of all capripoxviruses; however, further investigations are required.

The innovation of therapeutic modalities with better clinical efficacy is necessary for the treatment of patients with advanced cancers. Newcastle disease virus (NDV), an avian pathogenic virus, is one of the most promising oncolytic viruses that can replicate selectively in human cancer cells. In humans, NDV can cause transient conjunctivitis and mild flu-like symptoms. However, this virus poses no hazard to human health. The elucidation of the mechanisms of cancer cell killing by NDV is helpful for the clinical application of NDV in cancer patients. Regarding this, the present study was performed to evaluate apoptosis induction by NDV LaSota strain vaccine in human breast carcinoma cells. To this end, MCF-7 cells, a human breast adenocarcinoma cell line, were infected with NDV in vitro. Tumor cell cytotoxicity, apoptosis induction, and expression levels of apoptosis-related genes were examined in NDV-infected breast carcinoma cells. Tumor cell cytotoxicity was measured by 3-[4,5-dimethylthiazol-2yl]2,5-diphenyl-tetrazoliumbromide (MTT) assay. The induction of apoptosis was assessed by annexin V/propidium iodide staining. In addition, the expression levels of apoptosis-related genes were evaluated using real-time reverse transcription polymerase chain reaction (PCR) technique. The NDV showed cytotoxic effects on MCF-7 cells and induced apoptosis in the infected carcinoma cells. The gene expression levels of BAX, caspase-9, and caspase-3, but not BAK-1, were increased in NDV-infected cancer cells, compared to the gene expression levels in the non-infected cancer cells. These results suggest that the induction of the intrinsic pathway of apoptosis is one of the mechanisms that can contribute to cancer cell killing by NDV. Additional studies are required to investigate other probable mechanisms involved in NDV-mediated cancer cell killing.