Identification of a novel lineage of Crimean-Congo haemorrhagic fever virus in dromedary camels, United Arab Emirates.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne virus causing Crimean-Congo haemorrhagic fever (CCHF), a disease reported to have a high fatality rate in numerous countries. The virus is geographically widespread due to its vector, and numerous wild and domestic animals can develop asymptomatic infection. Serological and limited molecular evidence of CCHFV has previously been reported in Camelus dromedarius (the dromedary, or one-humped camel) in the United Arab Emirates (UAE). In this study, 238 camel samples were screened for CCHFV RNA where 16 camel samples were positive for CCHFV by RT-PCR. Analysis of full-length CCHFV genome sequences revealed a novel lineage in camels from the UAE, and potential reassortment of the M segment of the genome.

Contribution of glia cells specifically astrocytes in the pathology of depression: immunohistochemical study in different brain areas.

BACKGROUND: The contribution of glial cells to the pathophysiology of depression is an emerging research purpose. This study investigated the deficits in glial cells, specifically astrocytes in various brain regions, after the development of depression and then after voluntary running in depressed rats. MATERIALS AND METHODS: Forty-five adult male Wistar rats aged 8-10 weeks were used in the study. A depression model was generated through a forced swimming programme; voluntary running was allowed on rat running wheels; and brain sections were taken from the hippocampus, dentate gyrus (DG), medial prefrontal cortex (mPFC) and cerebellar cortex. After immunostaining with specific antibodies immuno-stain, the astrocytes, oligodendroglia and microglial cells were counted, and certain indices relating astrocytes to other glial cells were calculated. Astrocytic morphology was studied, and the optical density (OD) of glial fibrillary acidic protein (GFAP) immuno-expression was measured in different groups. RESULTS: Compared to the control group, animals in the depression group exhibited significant decreases in the mean astrocyte count in all studied brain areas, significant decreases in GFAP OD values in all areas and significantly reduced values for all glial astrocyte indices in the hippocampus, DG and mPFC. Compared to the rats in the depression group, those in the depression/exercise group exhibited significantly elevated mean astrocyte and oligodendroglia counts in all studied areas, significantly elevated GFAP OD values in all studied areas, and non-significant differences in glial astrocyte indices in the hippocampus, mPFC and cerebellar cortex. CONCLUSION: Astrocytes, rather than other glia, constitute a basis for the development and/or relief of depression.

Development of oriC-plasmids for use in Mycoplasma hyorhinis.

Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pig pathogen, belonging to the class Mollicutes. It causes polyserositis, arthritis and cancers in vitro, increasing attention of the researchers. Currently, there is no available genetic tool to manipulate its genome. This study describes a development of oriC-plasmids harboring either large (pGEMT-LoriC) or minimum (pGEMT-MoriC) origin of replication (oriC) of M. hyorhinis along with tetracycline resistance marker.These plasmids were successfully transformed into M. hyorhinis with average transformation frequency of 1.5 × 10-4 and 2.0 × 10-5 transformants/CFU for pGEMT-LoriC and pGEMT-MoriC respectively, and were integrated at the chromosomal oriC as well as remained freely replicating. We also constructed a Mini-oriC-HT1 targeting plasmid by inclusion of hlyC arms and was used to inactivate hlyC at average frequency of 50%. The efficiency of hlyC inactivation was further improved (by 90%) when Mini-oriC-HT2 that contains E. coli recA was used. In both cases, hemolysin mutant bacteria diminished the ability to lyse mouse RBCs compared to wild-type (P < 0.001). OriC-plasmids described in this study may, therefore open the way for functional genomics in M. hyorhinis. Furthermore, this is a first study demonstrated the gene associated with a hemolytic phenotype in mycoplasmas.

E. coli recA gene improves gene targeted homologous recombination in Mycoplasma hyorhinis.

Mycoplasma hyorhinis is an opportunistic pathogen of pigs. Recently, it has been shown to transform cell cultures, increasing the attention of the researchers. Studies on the pathogenesis require specific genetic tool that is not yet available for the pathogen. To address this limitation, we constructed two suicide plasmids pGEMT-tetM/LR and pGEMT-recA-tetM/LR having a tetracycline resistance marker flanked by two hemolysin gene arms. The latter plasmid encodes an E. coli recA, a gene involved in DNA recombination, repair and maintenance of DNA. Using inactivation of the hemolysin gene, which results in a detectable and measurable phenotype, we found that each plasmid can disrupt the hemolysin gene of M. hyorhinis through a double cross-over homologous recombination. However, inclusion of the E. coli recA gene in the construct resulted in 9-fold increase in the frequency of hemolysin gene mutants among the screened tetracycline resistance colonies. The resultant hemolysin mutant strain lacks the ability to lyse mouse bed blood cells (RBC) when tested in vitro (p<0.001). The host-plasmid system described in this study, has applications for the genetic manipulation of this pathogen and potentially other mycoplasmas.

GFP as a marker for transient gene transfer and expression in Mycoplasma hyorhinis.

Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pathogen of pigs and has been shown to transform cell cultures, which has increased the interest of researchers. The green florescence proteins (GFP) gene of Aquorea victoria, proved to be a vital marker to identify transformed cells in mixed populations. Use of GFP to observe gene transfer and expression in M. hyorhinis (strain HUB-1) has not been described. We have constructed a pMD18-O/MHRgfp plasmid containing the p97 gene promoter, origin of replication, tetracycline resistance marker and GFP gene controlled by the p97 gene promoter. The plasmid transformed into M. hyorhinis with a frequency of ~4 × 10(-3) cfu/µg plasmid DNA and could be detected by PCR amplification of the GFP gene from the total DNA of the transformant mycoplasmas. Analysis of a single clone grown on KM2-Agar containing tetracycline, showed a green fluorescence color. Conclusively, this report suggests the usefulness of GFP to monitor transient gene transfer and expression in M. hyorhinis, eventually minimizing screening procedures for gene transfer and expression.

In vitro protective efficacy of Lithium chloride against Mycoplasma hyopneumoniae infection.

Mycoplasma hyopneumoniae (M. hyopneumoniae) infection affects the swine industry. Lithium chloride (LiCl), is a drug used to treat bipolar disorder and has also shown activity against bacterial and viral infections. Herein, we evaluated the antibacterial activity of LiCl on PK-15 cells infected with M. hyopneumoniae. Incubation of LiCl (40mM) with cells for 24h, did not significantly affect the cell viability. The qRT-PCR showed ~80% reduction in M. hyopneumoniae genome when LiCl added post-infection. A direct effect of LiCl on bacteria was also observed. However, treatment of cells with LiCl prior infection, does not protect against the infection. Anti-bacterial activity of LiCl was further confirmed by IFA, which demonstrated a reduction in the bacterial protein. With 40mM LiCI, the apoptotic cell death, production of nitric oxide and superoxide anion induced by M. hyopneumoniae, were prevented by ~80%, 60% and 58% respectively. Moreover, caspase-3 activity was also reduced (82%) in cells treated with 40mM LiCl. LiCl showed activity against various strains of M. hyopneumoniae examined in our study. Collectively, our data showed that LiCl inhibited the infection of M. hyopneumoniae through anti-apoptotic mechanism.

Griffithsin binds to the glycosylated proteins (E and prM) of Japanese encephalitis virus and inhibit its infection.

Griffithsin (GRFT) is a broad-spectrum antiviral protein against several glycosylated viruses. In our previous publication, we have shown that GRFT exerted antiviral activity against Japanese encephalitis virus (JEV) infection. Herein, we further elucidated the mechanism by which GRFT inhibits JEV infection in BHK-21 cells. In vitro experiments using Pull-down assay and Co-immunoprecipitation (CO-IP) assay showed that GRFT binds to the JEV glycosylated viral proteins, specifically the enveloped (E) and premature (prM) glycoproteins. The binding of GRFT to the JEV was competitively inhibited by increasing concentrations of mannose; in turns abolished anti-JEV activity of GRFT. We suggested that, the binding of GRFT to the glycosylated viral proteins may contribute to its anti-JEV activity. Collectively, our data indicated a possible mechanism by which GRFT exerted its anti-JEV activity. This observation suggests GRFT's potentials in the development of therapeutics against JEV or other flavivirus infection.

Griffithsin inhibits Japanese encephalitis virus infection in vitro and in vivo.

Griffithsin (GRFT) is a broad-spectrum antiviral protein that is effective against several glycosylated viruses. Here, we have evaluated the in vitro and in vivo antiviral activities of GRFT against Japanese encephalitis virus (JEV) infection. In vitro experiments showed that treatment of JEV with GRFT before inoculation of BHK-21 cells inhibited infection in a dose-dependent manner, with 99 % inhibition at 100 µg/ml and a 50 % inhibitory concentration (IC(50)) of 265 ng/ml (20 nM). Binding assays suggested that binding of GRFT to JEV virions inhibited JEV infection. In vivo experiment showed that GRFT (5 mg/kg) administered intraperitoneally before virus infection could completely prevent mortality in mice challenged intraperitoneally with a lethal dose of JEV. Our study also suggested that GRFT prevents JEV infection at the entry phase by targeting the virus. Collectively, our data demonstrate that GRFT is an antiviral agent with potential application in the development of therapeutics against JEV or other flavivirus infections.

Inhibition of Japanese encephalitis virus infection in vitro and in vivo by pokeweed antiviral protein.

Pokeweed antiviral protein (PAP) is a plant-derived N-glycosidase ribosomal-inactivating protein isolated from Phytolacca americana. The antiviral activity of PAP has been described in several viruses. This study was to investigate the antiviral activity of PAP against Japanese encephalitis virus (JEV) infection in vitro and in vivo. Antiviral activity of PAP against JEV infection was evaluated in vitro using plaque forming assay, qRT-PCR and Western blot analysis. In vitro results showed that PAP inhibited replication of JEV in a dose-dependent manner with 50% inhibitory concentration (IC(50)) of 300 ng/ml (23.1 nM). Depurination assay suggested that the antiviral activity of PAP against JEV infection might be partially due to depurination of JEV genomic RNA. In vivo studies showed that PAP (1.0mg/kg) administered intraperitoneally decreased infection in mice challenged with a lethal dose of JEV, presenting a survival of 87.5% or 85.7% when administered pre-infection or post-infection. Collectively, our studies demonstrated that PAP possesses antiviral activity against JEV infection in vitro and in vivo, providing evidences for further development of PAP as an antiviral agent against JEV infection.

Oltipraz treatment in experimental schistosomiasis mansoni:VII. Effect of high dose oral therapy on alkaline phosphatase level in livers of infected mice.

Biochemical estimation of alkaline phosphatase (AP) in livers of mice experimentally infected with the Egyptian strain of S. mansoni before and after oltipraz treatment was carried out. The drug was given in a single oral dose of 500 mg/kg body weight. The results revealed that the drug led to gradual reduction in the AP mean level after 20 and 30 days from treatment. This was then followed by rise in the enzyme mean level, after 60 days. The drug also caused significant reduction in the AP mean level in livers of the drug control group of mice after 5 and 20 days from oltipraz administration in comparison normal uninfected controls. These findings might be attributed to a hepatocytotoxic effect of the drug.

Oltipraz treatment in experimental schistosomiasis mansoni: VI. Effect of high dose oral therapy on alkaline phosphatase level in the intestines of infected mice.

Extraction of alkaline phosphatase (AP) from the tissues of small intestines of mice experimentally infected with the Egyptian strain of S. mansoni before and after oltipraz treatment was carried out. The drug was given in a single oral dose of 500 mg/kg body weight. The results obtained revealed that the drug led to significant reduction in the AP mean level after 30 and 60 days from therapy in comparison to the infected untreated controls. Oltipraz also caused significant reduction in the intestinal AP mean level of the drug control group of mice after 5 and 20 days from drug administration in comparison to normal controls, which might be attributed to the toxic effect of the drug on the intestinal cells.

Oltipraz treatment in experimental schistosomiasis mansoni. II. Effect of high dose oral therapy on worm load and process of copulation in infected mice.

A single oral dose of 500 mg/kg body weight of Oltipraz was given to mice experimentally infected with the Egyptian strain of S. mansoni. The drug caused significant reduction in the number of worms and marked reduction in the percentage of paired worms in comparison to the infected untreated controls. This reduction in number was apparent after 5 days, 7 days and 2 days from Oltipraz administration, then the number gradually increased with elapsed time till the 60th day after therapy (the end of the work). The previous findings might indicate development of resistance to Oltipraz among the Egyptian strain of S. mansoni worms with prolongation the period of worms exposure to the drug.

Oltipraz treatment in experimental schistosomiasis mansoni: III. Effect of high dose oral therapy on hepatic shift of worms and tissue bound ova pattern in infected mice.

Oltipraz was given in a single oral dose of 500 mg per kg body weight to mice experimentally infected with the Egyptian strain of S. mansoni. The drug caused marked hepatic shift of the worms in comparison to the infected untreated controls. Hepatic shift of worms was apparent till the 30th. day from oltipraz administration, while pronounced back shift to mesenteric veins by the surviving parasites occurred 60 days post-treatment. The drug also caused death of all bilharzial eggs located inside tissues of the small intestines of infected mice and complete eradication of faecal eggs after the 5th., 7th., 20th., 30th. and 60th. days from therapy. These findings are in need of histopathological investigations.

Oltipraz treatment in experimental schistosomiasis mansoni. I. Morphological changes among S. mansoni worms induced by high dose oral therapy in mice.

Worms recovered from Oltiparz treated group of mice (500 mg/kg. body weight) show, suntedness, absent or poorly developed tuberculations, destroyed or poorly developed suckers, absence or severe atrophy of testes, complete absence or severe atrophy of ovaries, small or empty vitelline follicles and slightly or not pigmented intestines as compared with controls; the female worms are more affected than males. The morphological changes are apparent after 5 days, 7 days, 20 days and 30 days from drug administration, but are reversible in some surviving worms especially male worms after 60 days from therapy. The previous findings indicate that the drug causes severe degenerative changes in the structure of both S. mansoni male and female worms and that resistance to the drug develops among the Egyptian strain of S. mansoni worms with prolongation the period of worms exposure to the drug. The present results are discussed with those of others with some recommendations.

Further studies on the effect of niridazole on urinary and biliary excretion of copper.

Administration of niridazole to rats poisoned with copper caused a significant increase in both the urinary and biliary excretion of the metal. Although the urinary excretion of iron was increased by the drug, iron excretion was significantly decreased during the drug-induced excretion of copper after copper poisoning. Formation of a copper-niridazole chelate or chelates before excretion in the bile or urine may explain these findings. Polarity and molecular weights of the metal-drug chelates formed in vivo may be the directing forces not only in the selection of the metal for chelation, but also for its urinary or biliary excretion. The laboratory preparation of two copper-niridazole complexes lends support to these conclusions.