Molecular Characteristics and Pathogenicity of a Novel Transplacental Rat Cytomegalovirus

Cytomegalovirus (CMV) is a species-specific betaherpesvirus which causes acute, persistent and latent infections in both humans and animals. CMV is the most frequent congenital infection in humans. RCMV strain ALL-03 was the first CMV ever isolated from the placenta and uterus of the house rat (Rattus rattus diardii). As such, hypothetically, this RCMV should be a distinct strain from the existing isolates that is capable to cross placenta and infect the fetus. The objectives of the study were (i) to identify the novelty of the RCMV strain ALL-03, (ii) to characterize its immediateearly (IE) genes, and (iii) to determine its pathogenicity by developing the in utero transmission and neonatal infection models in rats. Overall, the present study signifies the virological and molecular detection of the RCMV antigen, DNA and mRNA in addition to the serological demonstration of the RCMV-specific immune response. Other than the traditional diagnostic methods, the study had also used advanced techniques, for examples, double antibody sandwich enzyme-linked immunosorbent iv assay (DAS-ELISA), quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. The study was commenced by characterizing the strain ALL-03. Upon infection, the virus showed delayed cytopathology, cellassociation, low maximum titres, the presence of herpesviral inclusion bodies and herpesvirus related particles in infected rat embryonic fibroblast (REF) cells; specific antigen-antibody reaction with RCMV strain Maastricht; and rat-specific are all in accord with a RCMV. The genetic difference at the genome level with that of Maastricht, English, UPM/Sg and UPM/Kn strains had confirmed its novelty. The first recognized genes expressed during CMV infection, the IE genes were studied by analyzing the mRNA transcripts of infected-REF cells. The cDNA libraries were cloned into plasmids for sequencing. Each sequence was then probed towards the databanks for an identity search. Following the PCR and hybridization techniques, two distinct transcripts of unknown identities within the databanks were confirmed to be of the strain ALL-03 origin. These two IE transcripts were found considerably different to the IE genes of RCMV strains Maastricht and English. Meanwhile, a real-time RT-PCR assay was developed specifically to quantify the in vitro transcription levels of the two RCMV IE mRNAs. The kinetic transcription profiles and the bioinformatics analyses suggested them as exon 4 or IE1 and exon 5 or IE2. An in utero infection model demonstrated the clinical signs, pathological changes and anatomical virus distribution to the uterus, placenta, embryo, fetus, lung, kidney, spleen, liver and salivary gland of rats. The placenta was observed to be involved in the maternofetal RCMV infection. The maternal viremia leading to uterine infection which subsequently transmitting to the fetus through the placenta is the most likely phenomenon of congenital CMV v infection in the model. The study has established a useful rat model that mimics the neonatal CMV infection in humans especially for the virus dissemination in different organs, viremia and immune response. The kinetic quantitation of the viral antigen, DNA and antibody was assessed by DAS-ELISA, real-time PCR and ELISA respectively. This neonatal rat model demonstrated a characteristic splenomegaly and acute virus dissemination in blood, spleen, liver, lung and kidney. The salivary gland infection is suggested to augment the antibody response that may be responsible for a reduction of viremia. The study has provided important new insights of CMV disease particularly for a congenital infection in humans. The exploitation of the major IE regions has permitted greatest advances as a candidate of viral-vectored immunocontraception for rat control and generation of eukaryotic expression vectors

Pathogenesis and vertical transmission of a transplacental rat cytomegalovirus

BACKGROUND: Cytomegalovirus (CMV) congenital infection is the major viral cause of well-documented birth defects in human. Because CMV is species-specific, the main obstacle to developing animal models for congenital infection is the difference in placental architecture, which preludes virus transmission across the placenta. The rat placenta, resembling histologically to that of human, could therefore facilitate the study of CMV congenital infection in human. RESULTS: In this report, we present clear evidences of the transplacental property of a new rat CMV (RCMV), namely ALL-03, which had been isolated from placenta and uterus of the house rat. Our study signifies the detection of infectious virus, virus particles, viral protein and DNA as well as immune response to demonstrate a natural model of acute CMV infection including the immunocompetent and immunocompromised host associated with or without pregnancy. It is characterized by a full range of CMV related clinical signs; lesions and anatomical virus distribution to uterus, placenta, embryo, fetus, neonate, lung, kidney, spleen, liver and salivary gland of the infected rats in addition to the virus-specific seroconversion. The preference of the virus for different organs mimics the situation in immunocompromised man. Most interestingly, the placenta was observed to be involved in the maternofetal infection and hence confirmed the hypothesis that the RCMV strain ALL-03 is capable to cross the placenta and infect the offsprings congenitally. CONCLUSION: The maternal viremia leading to uterine infection which subsequently infecting to the fetus through the placenta is the most likely phenomenon of CMV vertical transmission in our study

Cytotoxicity and apoptotic activities of alpha-, gamma- and delta-tocotrienol isomers on human cancer cells

Background: Tocotrienols, especially the gamma isomer was discovered to possess cytotoxic effects associated with the induction of apoptosis in numerous cancers. Individual tocotrienol isomers are believed to induce dissimilar apoptotic mechanisms in different cancer types. This study was aimed to compare the cytotoxic potency of alpha-, gamma- and delta-tocotrienols, and to explore their resultant apoptotic mechanisms in human lung adenocarcinoma A549 and glioblastoma U87MG cells which are scarcely researched. Methods: The cytotoxic effects of alpha-, gamma- and delta-tocotrienols in both A549 and U87MG cancer cells were first determined at the cell viability and morphological aspects. DNA damage types were then identified by comet assay and flow cytometric study was carried out to support the incidence of apoptosis. The involvements of caspase-8, Bid, Bax and mitochondrial membrane permeability (MMP) in the execution of apoptosis were further expounded. Results: All tocotrienols inhibited the growth of A549 and U87MG cancer cells in a concentration- and time-dependent manner. These treated cancer cells demonstrated some hallmarks of apoptotic morphologies, apoptosis was further confirmed by cell accumulation at the pre-G1 stage. All tocotrienols induced only double strand DNA breaks (DSBs) and no single strand DNA breaks (SSBs) in both treated cancer cells. Activation of caspase-8 leading to increased levels of Bid and Bax as well as cytochrome c release attributed by the disruption of mitochondrial membrane permeability in both A549 and U87MG cells were evident. Conclusions: This study has shown that delta-tocotrienol, in all experimental approaches, possessed a higher efficacy (shorter induction period) and effectiveness (higher induction rate) in the execution of apoptosis in both A549 and U87MG cancer cells as compared to alpha- and gamma-tocotrienols. Tocotrienols in particular the delta isomer can be an alternative chemotherapeutic agent for treating lung and brain cancers

Acalypha wilkesiana ethyl acetate extract enhances the in vitro cytotoxic effects of α-tocopherol in human brain and lung cancer cells

Multi-combinatorial approachesare considered nowadays to enhance the effectiveness of cancer treatment. In this study, α-tocopherol was tested in combination with the ethyl acetate extract from Acalypha wilkesiana for cytotoxicity activity against U87MG and A549 cell lines. The GI50 values for α-tocopherol against U87MG and A549 cells were 0.923±0.411μg/ml and 5.290±1.952μg/ml respectively in cell viability tests; when A. wilkesiana extract was added in adjunct with the treatment of α-tocopherol in minimum inhibitory concentration (MIC), the GI50 values of α-tocopherol improved significantly (p<0.05) to <0.43μg/ml (1μM) for both cell lines tested. Histological staining signified that both α-tocopherol and A. wilkesiana extract treated cancer cell lines exhibited apoptotic morphological characteristics. Single cell gel electrophoresis (SCGE) comet assays revealed that α-tocopherol caused only double strand DNA breaks; whereas A. wilkesiana extract caused both single strand and double strand DNA breaks in U87MG and A549 cells. It is proposed that α-tocopherol and A. wilkesiana extract might trigger apoptosis in both U87MG and A549 cells through different apoptotic pathways that might complement each other to enhance their antiproliferative efficacy against the cancer cells

Engineering of Thermovibrio ammonificans carbonic anhydrase mutants with increased thermostability

Carbonic anhydrase can be used as an additive to improve the efficiency of carbon capture and utilisation processes, due to its ability to increase the rate of CO2 absorption into solvents. Successful industrial application requires robust carbonic anhydrases, able to withstand process conditions and to perform consistently over long periods of time. Tolerance of high temperatures, pH and salt concentrations are particularly desirable features. We have previously used molecular dynamics simulations to rationally design four mutants of Thermovibrio ammonificans carbonic anhydrase with increased rigidity, and we hypothesized that this will result in an increased thermostability. Herein, we report on the successful recombinant expression and characterization of these mutants. Four of the TaCA variants showed increased stability at 90 ᵒC during 1 h, compared to wild-type. Two out of the four mutations predicted by the theoretical studies resulted in marked stabilization of the protein, with up to 3-fold higher time of half-life for mutant N140 G compared to the wild-type enzyme at 60 ᵒC. A significantly 50-fold increased ester hydrolysis activity was also observed with the most thermostable variant at 95 ᵒC compared to 25 ᵒC, suggesting an increased flexibility of the active site at high temperatures

Molecular characterisation and identification of the major immediate-early (MIE) genes of rat cytomegalovirus (RCMV)

The paper first describes the characterisation and identification of two transcripts of major immediate-early (MIE) genes of rat cytomegalovirus (RCMV) strain ALL-03 using molecular approach. In the absence of any known sequences in the strain ALL-03, IE genes, the first recognised viral genes expressed during infection were the most important genetic source for the exploration of the viral genome. The steps taken were the construction of cDNA libraries from rat embryonic fibrolast (REF) infected with RCMV strain ALL-03 under 'IE' condition by using several techniques such as RT-PCR, PCR, cloning, sequencing and bioinformatics. Two transcripts were confirmed to be of RCMV strain ALL-03 origin by specific PCR and hybridisation analyses. Based on the bioinformatics analyses, it is most likely, that these transcripts are exon 4 (IE1) and exon 5 (IE2) genes. The considerable divergence in the nucleotide sequence of the two IE transcripts from those of MIE regions of both Maastricht and English strains further supports the claim that the ALL-03 is a distinct RCMV strain indeed

Quantitative approach in screening the antiviral properties of Kandis Hutan in animal cell culture

This main aim of this study was to determine the concentration of Kandis Hutan leaf extracts that can inhibit the infectivity of pseudorabies virus (PrV) in Vero cells. The leaf extracts were crude and extracted with 3 organic solvents namely hexane, ethyl acetate and ethanol. The cytotoxic effect of extracts on Vero cells was assessed by both MTT assay and cell cytotoxicity scoring method. Two-fold serial dilutions of each extracts were prepared from the highest concentration of 1000µg/ml in 0.1% DMSO. For MTT assay, the highest cytotoxicity was found in the hexane extract (CC50 <1.25 µg/mL), followed by ethyl acetate extract (CC50 = 237.5 µg/mL), whilst minimal cell cytotoxicity was observed in ethanol extracts (CC50 = 555.0 µg/mL). There was a significant correlation between cell scoring system and MTT assay in term of cell cytotoxicity whereby the least toxic was ethanol extracts, followed by ethyl acetate extract and the most toxic hexane extract. Four antiviral assays were conducted for each extracts, namely plaque reduction assay, cytopathic effect (CPE) reduction assay, inhibition assay and virucidal assay. The most promising result was obtained from the inhibition assay, in which ethyl acetate extracts produced 75% viral inhibition at 125 µg/mL concentration. In virucidal assay, both ethyl acetate and ethanol extracts produced 100% viral inhibition at 250 µg/mL. For plaque reduction assay, there was a significant dose dependent inhibition for ethyl acetate extract but not for ethanol extract and hexane extract. In comparison to CPE reduction assay, findings from plaque reduction assay showed better viral inhibition by ethyl acetate extract (47%) at concentration of 300 µg/mL. The estimated selectivity index (ESI) calculated from the inhibition assay showed the highest antiviral response by ethyl acetate extract (2.7) in comparison with ethanol extract (1.8) and hexane extract (0.1). Therefore, the most promising antiviral activity was produced by ethyl acetate extract which showed consistent viral inhibition in all tested antiviral assays. In contrary, hexane extracts showed the least antiviral efficacy among the tested extracts

Quantitative in vitro assays for screening antiviral characteristics of Berembang Bukit leaf extracts

Berembang Bukit or Megawasih, is a native of our tropical rainforest and other ASEAN countries. In a preliminary review, a qualitative screening on the antiviral activities of its leaf extracts revealed various degrees of antiviral potential. Therefore this study was done to evaluate its antiviral characteristics in a quantitative approach. The leaf extracts were prepared using hexane, ethyl acetate and ethanol extractions and dissolved in DMSO. DMSO cytotoxicity was initially evaluated to ensure a safe working concentration. A negligible cytotoxicity was observed at a concentration of ≤ 0.1% DMSO. The cytotoxic effect of extracts on Vero cells was assessed by both MTT assay and cell cytotoxicity scoring method. Two-fold serial dilutions of each extracts were prepared from the highest concentration of 1000µg/mL in 0.1% DMSO. For MTT assay, the highest cytotoxicity was found in the ethyl acetate extract (CC50 = 218µg/mL), whilst minimal cell cytotoxicity was observed in both hexane (CC50 = 833µg/ml) and ethanol (CC50 = >1000µg/mL) extracts. However, there were no correlation between MTT and cell scoring for cytotoxicity in this study. A series of experiments including CPE reduction assay, plaque reduction assay, inhibition assay and virucidal assay were done to evaluate the total antiviral potential of the leaf extracts. The leaf extracts produced a dose-dependent antiviral response. Both ethyl acetate and ethanol extracts showed 100% plaque formation inhibition in plaque reduction assay, inhibition assay and virucidal assay. Hexane extracts showed absence of plaque inhibition in all the tested antiviral assays. In inhibition assay, the estimated selective index (ESI) for ethanol and ethyl acetate extracts were 8.3 and 1.9, respectively. Whilst in CPE reduction assay, the ESI for the respective extracts were 6.7 and 2.9. In conclusion, the ethanol extracts exhibited the highest antiviral efficacy among the tested extracts

Antiviral properties of Berembang Bukit and Kandis Hutan against psedorabies virus in animal cell culture

Tropical rainforest in Malaysia represents and untapped potential source of antiviral compounds. Bioactive compounds in plant species from the same genus as Kandis Hutan such as xanthones, benzophenones, biflavonoids and lupeol had been studied. Eugeniin is an anti-herpesvirus compound which had also been found n Berembang Bukit. This preliminary study was carried out to discover the presence of antiviral properties in Berembang Bukit and Kandis Hutan using different antiviral assays. In this study, MTT cell viability assay was used in addition to microscopic evaluation of pseudorabies virus (PrV)- induced cytopathic effects (CPE) on Vero cells. The cellular toxicity of DMSO was also evaluated. DMSO was less than 10% cytotoxic at concentration of 0.1% to Vero cells and its effect can be negligible. Both plants had demonstrated antiviral properties in thyl acetate and ethanol extracts. From our findings from all three antiviral assays, the ethanol-extracted Kandis Hutan possessed the most promising antiviral properties. Nevertheless, antiviral potential of ethyl acetate and ethanol-extracted Berembang Bukit and ethyl acetate-extracted Kandis Hutan also merit further investigatio