Infected Vero Cells Research Articles

No evidence of SARS-CoV-2 RNA among blood donors: A multicenter study in Hubei, China.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) RNA could be detected in the blood of infected cases. From February 9, all blood establishments in Hubei province, China, implemented nucleic acid testing (NAT) for SARS‐CoV‐2 RNA among blood donors to ensure blood safety.Study Design and MethodsNucleic acid test screening individually (ID) or by minipool (MP) testing was performed according to the manufacturerʼs instructions. Inactivated culture supernatant of SARS‐CoV‐2–infected Vero cells was quantified by droplet digital polymerase chain reaction (ddPCR) and series diluted with negative plasma to evaluate the assayʼs performance.ResultsThe limit of detection of the kit for MP testing was 62.94 and 33.14 copies/mL for N and ORF1ab region, respectively. ID testing could achieve 3.87 and 4.85 copies/mL for two regions using 1600 μL of plasma. Coefficients of variations of two different concentrations of reference samples were all less than 5% in MP testing. As of April 30, 2020, a total of 98,342 blood donations including 87,095 whole blood donations and 11,247 platelet donations were tested by ID or MP testing, and no RNAemia was found. In addition, Hubei province suffered precipitously decreased blood supply, especially in February: 86% reduction compared with the same period of 2019.ConclusionNucleic acid test screening of SARS‐CoV‐2 on blood donations is suitable in blood establishments using the commercial real‐time PCR detection kit based on available instruments. The negative result indicated that SARS‐CoV‐2 appears to be no direct threat to blood safety but raises some serious issues for general blood supply.

Heat Shock Protein Member 8 Is an Attachment Factor for Infectious Bronchitis Virus.

Although infectious bronchitis virus (IBV) is the first coronavirus identified, little is known about which membrane protein of host cells could interact with IBV spike protein and facilitate the infection by the virus. In this study, by using a monoclonal antibody to the S1 protein of IBV M41 strain, we found that heat shock protein member 8 (HSPA8) could interact with spike protein of IBV. HSPA8 was found to be present on the cell membrane and chicken tissues, with highest expression level in the kidney. Results of co-IP and GST-pull-down assays indicated that the receptor binding domain (RBD) of IBV M41 could interact with HSPA8. The results of binding blocking assay and infection inhibition assay showed that recombinant protein HSPA8 and antibody to HSPA8 could inhibit IBV M41 infection of chicken embryonic kidney (CEK) cells. Further, we found that HSPA8 interacted with the N-terminal 19–272 amino acids of S1 of IBV Beaudette, H120 and QX strains and HSPA8 from human and pig also interacted with IBV M41-RBD. Finally the results of binding blocking assay and infection inhibition assay showed that recombinant HSPA8 protein and antibody to HSPA8 could inhibit IBV Beaudette strain infection of Vero cells that were treated with heparanase to remove heparan sulfate from the cell surface. Taken together, our results indicate that HSPA8 is a novel host factor involved in IBV infection.

Shortlisting SARS-CoV-2 Peptides for Targeted Studies from Experimental Data-Dependent Acquisition Tandem Mass Spectrometry Data.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a crucial tool for fighting the COVID‐19 pandemic. This dataset brief presents the exploration of a shotgun proteomics dataset acquired on SARS‐CoV‐2 infected Vero cells. Proteins from inactivated virus samples were extracted, digested with trypsin, and the resulting peptides were identified by data‐dependent acquisition tandem mass spectrometry. The 101 peptides reporting for six viral proteins were specifically analyzed in terms of their analytical characteristics, species specificity and conservation, and their proneness to structural modifications. Based on these results, a shortlist of 14 peptides from the N, S, and M main structural proteins that could be used for targeted mass‐spectrometry method development and diagnostic of the new SARS‐CoV‐2 is proposed and the best candidates are commented.

Anti-Neospora caninum IgG in fetal bovine serum

Neospora caninum is an obligate intracellular protozoan, recognized as an important cause of abortion in infection cattle worldwide, and is responsible for major economic losses in beef and dairy herds. There are scarce data on the presence of antibodies in fetal serum samples from beef cattle. The pathogen is straggling mainly by vertical transmission in ruminants. This study aimed to determine the frequency of anti-N. caninum antibodies in fetal bovine serum. Overall, 751 fetal serum samples from slaughterhouses in Uruguaiana and Santo Ângelo (Rio Grande do Sul state) were used. Serologic diagnosis was made using indirect immunofluorescence assay (IFA). Slides were prepared by culturing infected Vero cells using tachyzoites of the N. caninum NC-1 strain and a serum dilution of 1:25. Of the 751 samples analyzed, 11.7% (88/751) were positive in the IFA assay. The results indicate an occurrence of neosporosis in the studied region, as well as the possibility of transplacental transmission of the protozoon, characterizing an important mechanism of propagation for the disease.

Genome-wide analysis of long noncoding RNA profiles in Vero cells infected with porcine epidemic diarrhea virus.

Porcine epidemic diarrhea (PED) is an acute enteric disease caused by porcine epidemic diarrhea virus (PEDV). In China, variant PEDV causes severe watery diarrhea, vomiting, and dehydration in piglets, leading to very high morbidity and mortality. However, the pathogenesis of PEDV is still not fully understood. In our study, we analyzed the long noncoding RNA (lncRNA) and mRNA expression profiles of PEDV GDgh16 in infected Vero cells at 60h postinfection. A total of 61,790 annotated mRNAs, 14,247 annotated lncRNAs and 1290 novel lncRNAs were identified. A total of 227 annotated lncRNAs and 13 novel lncRNAs were significantly and differentially expressed after viral infection. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases were used to identify genes adjacent to the lncRNAs, and it was found that these lncRNAs were enriched in pathways related to immune and antiviral responses. Next, we selected candidate lncRNAs and their predicted target genes for study. RT-qPCR demonstrated that these lncRNAs and genes were differentially expressed after PEDV infection. Our study investigated the function of lncRNAs involved in PEDV infection, providing new insight into the pathogenic mechanisms of PEDV.

Biochemical characterization and application of a novel lectin from the cyanobacterium Lyngabya confervoides MK012409 as an antiviral and anticancer agent

In the present study, a novel lectin was purified from the newly isolated cyanobacterium, Lyngabya confervoides MK012409 and tested for its antiviral and anticancer activity. Out of 30 isolates, Mabroka-s isolate which identified as Lyngabya confervoides MK012409 showed the highest agglutination titer. Lyngabyal lectin showed the greatest haemagglutination activity with pigeon/rabbit erythrocytes with a minimum concentration of 2.4 μg/ml. Physical characterization of Lyngabyal lectin showed ability to keep the activity at a higher temperature up to 80 °C with stability over a wide pH range (4–8) as well as its stability toward chemical denaturants. Carbohydrate specificity test revealed that the sugar alcohols completely inhibited the lectin haemagglutination activity. The electrophoretic analysis revealed that the lyngabyal lectin is a 140 kDa composed of two 70 kDa subunits. Lyngabyal lectin was able to inhibit the proliferation of MCF-7 and Caco-2 cancer cell lines with IC50 values of 246 ± 0.17 and 376.4 ± 0.34 μg/ml, respectively. Lyngabyal lectin also showed virucidal activity against HSV-1 with EC50 of 167 ± 0.52 ng/ml and inhibited plaque formation in the HSV-1 infected Vero cells with EC50 of 84.94 ± 0.34 ng/ml. These findings emphasize the ability of the lyngabyal lectin to fight breast and colon cancer besides it represents a promising antiviral agent.

Reagents for detection of Rift Valley fever virus infection in sheep.

Rift Valley fever virus (RVFV) causes Rift Valley fever (RVF), resulting in morbidity and mortality in humans and ruminants. Evidence of transboundary outbreaks means that RVFV remains a threat to human health and livestock industries in countries that are free from the disease. To enhance surveillance capability, methods for detection of RVFV are required. The generation of reagents suitable for the detection of RVFV antigen in formalin-fixed, paraffin-embedded tissues from infected animals have been developed and are described herein. Recombinant nucleoprotein (rNP) was expressed in Escherichia coli and purified using immobilized metal ion affinity chromatography. Purified rNP was used as an immunogen to produce anti-NP polyclonal antisera in rabbits for use in detection of RVFV NP in experimentally infected animals by immunohistochemistry. Antisera raised in rabbits against rNP were able to recognize viral NP antigen in fixed infected Vero cell pellets and sheep liver. Therefore, the methods and reagents described herein are useful in assays for detection of RVFV infections in animals, for research and surveillance purposes.

Single AAV-Mediated CRISPR-SaCas9 Inhibits HSV-1 Replication by Editing ICP4 in Trigeminal Ganglion Neurons

Herpes simplex keratitis (HSK) is the most common cause of corneal blindness in developed nations, caused by primary or recurrent herpes simplex virus 1 (HSV-1) infection of the cornea. Latent infection of HSV-1, especially in the trigeminal ganglion (TG), causes recurrence of HSV-1 infection. As antiviral treatment is not effective on latent HSV-1, to test the possibility of inhibiting HSV-1 by SpCas9 (Streptococcus pyogenes Cas9) or SaCas9 (Staphylococcus aureus Cas9), ICP0 and ICP4, two important genes required for HSV-1 replication and reactivation, were chosen as targets. In Vero cells, SpCas9 and SaCas9 targeting ICP0 or ICP4 can effectively inhibit the proliferation of HSV-1 without affecting cell viability. No significant guide RNA (gRNA)-dependent off-targets were observed in the human genome by digenome sequencing and deep sequencing verification. Adeno-associated virus 1 (AAV1)-mediated delivery of SaCas9 inhibits HSV-1 replication by targeting ICP4 in mouse primary TG neuronal cells. SpCas9 and SaCas9 are able to inhibit HSV-1 infection in Vero cells and mouse TG neuronal cultures with high efficiency and good biosafety. AAV1-mediated delivery of SaCas9 shows great potential in treating HSK and inhibiting HSV-1 in TG neurons. Further investigations may be needed to test the inhibition of latent infections, which may result in the development of novel methods for treating viral diseases.

Caspase-mediated cleavage of nucleocapsid protein of a protease-independent porcine epidemic diarrhea virus strain

Porcine epidemic diarrhea virus (PEDV) infection in neonatal piglets can cause up to 100% mortality, resulting in significant economic loss in the swine industry. Like other coronaviruses, PEDV N protein is a nucleocapsid protein and abundantly presents at all stages of infection. Previously, we reported that the N protein of trypsin-independent PEDV 8aa is cleaved during virus replication. In this study, we further investigated the nature of N protein cleavage using various methods including protease cleavage assays with or without various inhibitors and mutagenesis study. We found that PEDV 8aa infection in Vero cells leads to apoptotic cell death, and caspase 6 or 7 can cleave PEDV 8aa N protein at the late stage of the replication. The caspase-mediated cleavage occurs between D424 and G425 near the C-terminal of N protein. We also report that both cleaved and uncleaved N proteins are exclusively localized in the cytoplasm of PEDV infected cells.

Patient-Derived Mutations Impact Pathogenicity of SARS-CoV-2

The sudden outbreak of the severe acute respiratory syndrome–coronavirus (SARS-CoV-2) has spread globally with more than 1,300,000 patients diagnosed and a death toll of 70,000. Current genomic survey data suggest that single nucleotide variants (SNVs) are abundant. However, no mutation has been directly linked with functional changes in viral pathogenicity. We report functional characterizations of 11 patient-derived viral isolates. We observed diverse mutations in these viral isolates, including 6 different mutations in the spike glycoprotein (S protein), and 2 of which are different SNVs that led to the same missense mutation. Importantly, these viral isolates show significant variation in cytopathic effects and viral load, up to 270-fold differences, when infecting Vero-E6 cells. Therefore, we provide direct evidence that the SARS-CoV-2 has acquired mutations capable of substantially changing its pathogenicity. Funding: This work was supported by funds from Major Project of Zhejiang Provincial Science and Technology Department #2020C03123, National Science and Technology Major Project for the Control and Prevention of Major Infectious Diseases in China (2018ZX10711001, 2018ZX10102001, 2018ZX10302206), and start-606 up funds from Life Sciences Institute at Zhejiang University. Conflict of Interest: None. Ethical Approval: The study was approved by the Clinical Research Ethics Committee of The First Affiliated Hospital, School of Medicine, Zhejiang University (Approval notice 2020-29) for emerging infectious diseases.

Xanthenedione (and intermediates involved in their synthesis) inhibit Zika virus migration to the central nervous system in murine neonatal models

Zika Virus (ZIKV), an arbovirus that belongs to the Flaviviridae family, has become a global concern since its outbreak in the Americas in 2015. With symptoms similar to other Flavivirus as Dengue and Yellow Fever viruses, infections by ZIKV have also been related to several neurological complications such as microcephaly in newborns and Guillain-Barre syndrome. Considering the high prevalence of ZIKV infection in certain areas, the risks that the virus poses to fetal brain development, and the fact that there is no vaccine or specific prophylaxis available, an effective treatment capable of preventing the infection is of potential interest. Therefore, in the present investigation, the antiviral activity on ZIKV of a group of xanthenodiones and intermediate ketones involved in their synthesis was evaluated for the first time. It was found that the compound 2-(2,6-dichlorobenzylidene)cyclohexane-1,3-dione 27 was able to completely inhibit the viral infection of Vero cells as well as to significantly reduce viral load in the brains of newborn Swiss mice. These effects are related to a direct interaction of the compound with the viral particle, blocking the viral adsorption.

RETRACTED ARTICLE: EV71 virus reduces Nrf2 activation to promote production of reactive oxygen species in infected cells

BackgroundEmerging evidence closely links Enterovirus 71 (EV71) infection with the generation of reactive oxygen species (ROS). Excess ROS results in apoptosis and exacerbates inflammatory reactions. The Keap1–Nrf2 axis serves as an essential oxidant counteracting pathway.MethodsThe present study aimed to elucidate the role of the Keap1–Nrf2 pathway in modulating apoptosis and inflammatory reactions triggered by oxidative stress in Vero and RD cells upon EV71 infection.ResultsElevated ROS production was identified in EV71 infected Vero and RD cells. The percentage of dead cells and expression of inflammation-promoting cytokines were increased in these cells. EV71 infected cells also displayed reinforced Keap1 expression and abrogated Nrf2 expression. Keap1 silencing resulted in the downstream aggregation of the Nrf2 protein and heme oxygenase-1 HO-1. Keap1 silencing repressed ubiquitination and reinforced Nrf2 nuclear trafficking. Furthermore, silencing Keap1 expression repressed ROS production, cell death, and inflammatory reactions in EV71 infected RD and Vero cells. In contrast, silencing of both Keap1 and Nrf2 restored ROS production, cell death, and inflammatory reactions. Nrf2 and Keap1 modulated the stimulation of the Akt sensor and extrinsic as well as intrinsic cell death pathways, resulting in EV71-triggered cell death and inflammatory reactions.ConclusionsEV71 infection can trigger ROS production, cell death, and inflammatory reactions by modulating the Nrf2 and Keap1 levels of infected cells.

Vero Cells as a Mammalian Cell Substrate for Human Norovirus.

Human norovirus (HuNoV) is a principal cause of acute gastroenteritis worldwide, particularly in developing countries. Its global prevalence is underscored by more serious morbidity and some mortality in the young (<5 years) and the elderly. To date, there are no licensed vaccines or approved therapeutics for HuNoV, mostly because there are limited cell culture systems and small animal models available. Recently described cell culture systems are not ideal substrates for HuNoV vaccine development because they are not clonal or only support a single strain. In this study, we show Vero cell-based replication of two pandemic GII.4 HuNoV strains and one GII.3 strain and confirm exosome-mediated HuNoV infection in Vero cells. Lastly, we show that trypsin addition to virus cultures or disruption of Vero cell host genes can modestly increase HuNoV replication. These data provide support for Vero cells as a cell culture model for HuNoV.

Porcine Epidemic Diarrhoea Virus Induces Cell-cycle Arrest through the DNA Damage-signalling Pathway.

IntroductionPorcine epidemic diarrhoea virus (PEDV) infection causes watery diarrhoea, vomiting, anorexia, and weight loss, especially among neonatal piglets, inflicting on them morbidity and mortality potentially reaching 90%–100%. Despite it being known that certain mammalian cell phases are arrested by PEDV, the mechanisms have not been elucidated, and PEDV pathogenesis is poorly understood. This study determined the effect of an epidemic PEDV strain on cell cycle progression.Material and MethodsWe observed the effect of the PEDV SHpd/2012 strain on an infected Vero cell cycle through flow cytometry and Western blot, investigating the interrelationships of cell-cycle arrest, the DNA damage–signalling pathway caused by PEDV and the phosphorylation levels of the key molecules Chk.2 and H2A.X involved upstream and downstream in this pathway.ResultsPEDV induced Vero cell-cycle arrest at the G1/G0 phase. The phosphorylation levels of Chk.2 and H2A.X increased with the prolongation of PEDV infection, and no significant cell-cycle arrest was observed after treatment with ATM or Chk.2 inhibitors. The proliferation of PEDV was also inhibited by treatment with ATM or Chk.2 inhibitors.ConclusionPEDV-induced cell-cycle arrest is associated with activation of DNA damage–signalling pathways. Our findings elucidate the molecular basis of PEDV replication and provide evidence to support further evaluation of PEDV pathogenesis.

Examination of vesicular stomatitis virus-induced morphology changes in individual Vero cells by QMod microscopy.

Viral infection of cultured cells induces changes in the biophysical characteristics of the affected cells. Advanced microscopic cameras such as Ovizio's QMod, coupled with the appropriate software, can measure a variety of characteristics on a per-cell basis. We have employed this system to monitor the progression of vesicular stomatitis virus infection in Vero cells and to describe the cellular changes associated with advancing vesicular stomatitis virus infection. The measurements of cellular characteristics are operator-independent, and the goal is to establish a robust method to mathematically determine viral infection levels in a given sample. This will provide a means to measure viral titer in a faster and less subjective way than manual reading of plaque assays or tissue culture infectious dose 50 assays.

Knocking Down TcNTPDase-1 Gene Reduces in vitro Infectivity of Trypanosoma cruzi.

Ecto-Nucleoside Triphosphate Diphosphohydrolases are enzymes that hydrolyze tri- and/or diphosphate nucleosides. Evidences pointed out to their participation in Trypanosoma cruzi virulence, infectivity, and purine acquisition. In this study, recombinant T. cruzi knocking out or overexpressing the TcNTPDase-1 gene were built, and the role of TcNTPDase-1 in the in vitro interaction with VERO cells was investigated. Results show that epimastigote forms of hemi-knockout parasites showed about 50% lower level of TcNTPDase-1 gene expression when compared to the wild type, while the T. cruzi overexpressing this gene reach 20 times higher gene expression. In trypomastigote forms, the same decreasing in TcNTPDase-1 gene expression was observed to the hemi-knockout parasites. The in vitro infection assays showed a reduction to 51.6 and 59.9% at the adhesion and to 25.2 and 26.4% at the endocytic indexes to the parasites knockout to one or other allele (Hygro and Neo hemi-knockouts), respectively. In contrast, the infection assays with T. cruzi overexpressing TcNTPDase-1 from the WT or Neo hemi-knockout parasites showed an opposite result, with the increasing to 287.7 and 271.1% at the adhesion and to 220.4 and 186.7% at the endocytic indexes, respectively. The parasitic load estimated in infected VERO cells by quantitative real time PCR corroborated these findings. Taken together, the partial silencing and overexpression of the TcNTPDase-1 gene generated viable parasites with low and high infectivity rates, respectively, corroborating that the enzyme encoded for this gene plays an important role to the T. cruzi infectivity.

In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

BackgroundThe Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) first broke out in Wuhan (China) and subsequently spread worldwide. Chloroquine has been sporadically used in treating SARS-CoV-2 infection. Hydroxychloroquine shares the same mechanism of action as chloroquine, but its more tolerable safety profile makes it the preferred drug to treat malaria and autoimmune conditions. We propose that the immunomodulatory effect of hydroxychloroquine also may be useful in controlling the cytokine storm that occurs late-phase in critically ill SARS-CoV-2 infected patients. Currently, there is no evidence to support the use of hydroxychloroquine in SARS-CoV-2 infection.MethodsThe pharmacological activity of chloroquine and hydroxychloroquine was tested using SARS-CoV-2 infected Vero cells. Physiologically-based pharmacokinetic models (PBPK) were implemented for both drugs separately by integrating their in vitro data. Using the PBPK models, hydroxychloroquine concentrations in lung fluid were simulated under 5 different dosing regimens to explore the most effective regimen whilst considering the drug’s safety profile.ResultsHydroxychloroquine (EC50=0.72 μM) was found to be more potent than chloroquine (EC50=5.47 μM) in vitro. Based on PBPK models results, a loading dose of 400 mg twice daily of hydroxychloroquine sulfate given orally, followed by a maintenance dose of 200 mg given twice daily for 4 days is recommended for SARS-CoV-2 infection, as it reached three times the potency of chloroquine phosphate when given 500 mg twice daily 5 days in advance.ConclusionsHydroxychloroquine was found to be more potent than chloroquine to inhibit SARS-CoV-2 in vitro.

Susceptibility of Chicken Embryos, Sheep, Cattle, Pigs, and Chickens to Zika Virus Infection.

The susceptibility of sheep, cattle, pigs, chickens and chicken embryos to Zika virus infection was evaluated by experimental inoculation with Zika virus Thailand strain isolated from a Canadian traveler in 2013. The inoculated animals did not develop any clinical signs of disease nor evidence of Zika virus replication in peripheral blood, cerebrospinal fluid and tissues including brain and spinal cord assessed by real-time RT-PCR. Sera were also negative for Zika virus antibodies by Zika virus neutralization assays as well as Zika virus immunoperoxidase staining of Zika infected Vero cells. Chicken embryos were inoculated by different routes including yolk sac (4 day old embryos), chorioallantoic membrane (8 day old embryos), amniotic fluid (8 day old embryos) and intravenous routes (12 day old embryos). Virus replication in chicken embryos was observed in the brain and body tissues following intravenous (IV), yolk sac (YS), chorioallantoic membrane (CAM), and amniotic fluid (AF) inoculation routes. The highest mortality was observed in embryos inoculated via yolk sac. The dead embryos showed diffuse muscular hemorrhages. The yolk sac inoculated chicken embryos showed delayed hatching and displayed neurological signs immediately after hatching. These studies demonstrate that 8 week old sheep, 6 month old cattle, 4 week old pigs, and 4 week old chickens are not susceptible to Zika virus infection when inoculated experimentally and therefore unlikely to pose a risk as Zika virus reservoirs. However, chicken embryos are highly susceptible to Zika virus resulting in clinical disease of chicks after hatching. This study demonstrates that Zika virus has a tropism for embryonic tissue and that chicken embryos can be used as a model to study Zika virus replication and pathogenesis.

Virus goes viral: an educational kit for virology classes

BackgroundViruses are the most numerous entities on Earth and have also been central to many episodes in the history of humankind. As the study of viruses progresses further and further, there are several limitations in transferring this knowledge to undergraduate and high school students. This deficiency is due to the difficulty in designing hands-on lessons that allow students to better absorb content, given limited financial resources and facilities, as well as the difficulty of exploiting viral particles, due to their small dimensions. The development of tools for teaching virology is important to encourage educators to expand on the covered topics and connect them to recent findings. Discoveries, such as giant DNA viruses, have provided an opportunity to explore aspects of viral particles in ways never seen before. Coupling these novel findings with techniques already explored by classical virology, including visualization of cytopathic effects on permissive cells, may represent a new way for teaching virology. This work aimed to develop a slide microscope kit that explores giant virus particles and some aspects of animal virus interaction with cell lines, with the goal of providing an innovative approach to virology teaching.MethodsSlides were produced by staining, with crystal violet, purified giant viruses and BSC-40 and Vero cells infected with viruses of the genera Orthopoxvirus, Flavivirus, and Alphavirus. Slides with amoebae infected with different species of giant viruses and stained with hemacolor reagents were also produced.ResultsStaining of the giant viruses allowed better visualization of the viral particles, and this technique highlights the diversity in morphology and sizes among them. Hemacolor staining enabled visualization of viral factories in amoebae, and the staining of infected BSC-40 and Vero cell monolayers with crystal violet highlights plaque-forming units.ConclusionsThis kit was used in practical virology classes for the Biological Sciences course (UFMG, Brazil), and it will soon be made available at a low-cost for elementary school teachers in institutions that have microscopes. We hope this tool will foster an inspiring learning environment.

The effect of edelfosine on GRA1 and MIC3 expressions in acute toxoplasmosis.

Phosphoinositide-dependent phospholipase-C (PI-PLC) triggers the calcium signaling pathway which plays an important role in dense granule and microneme secretion and pathogenesis of Toxoplasma gondii (T. gondii). There are limited data about the effects of phospholipid analogues against T. gondii. The current study assessed the effect of edelfosine, as a phospholipid analogue, on GRA1 and MIC3 expressions using in vitro and in vivo models of acute toxoplasmosis. Infected Vero cells were treated by edelfosine in two subgroups: 24h following the cell infection and treatment at the same time of cell infection. Animal study was performed on forty mice in four groups including non-infected, infected untreated, infected edelfosine-treated, and infected pyrimethamine-treated. Gene and protein expression analyses were done using quantitative real-time PCR and western blot, respectively. Edelfosine significantly reduced the GRA1 (P < 0.01) and MIC3 (P < 0.01) mRNA and protein expressions in 24h following the cell infection and at the same time of cell infection groups. In vivo study showed that the edelfosine significantly reduced the GRA1 expression in eye, and MIC3 expression in brain and liver. Moreover, the edelfosine-treated infected mice had significant higher survival rate compared with uninfected mice. The reducing effect of edelfosine on GRA1 and MIC3 mRNA and protein levels 24h following the cell infection was more than treatment at the same time of cell infection group. Moreover, the effect of edelfosine on GRA1 and MIC3 expression in animal tissues was variable. These data showed that the edelfosine may decrease the T. gondii excretory/secretory antigens through inhibition of PI-PLC.