Development of a semi-quantitative real-time RT-PCR for the detection of measles virus

Comparative evaluation of new TaqMan real-time assays for the detection of hepatitis A virus

Evaluation of viral extraction methods on a broad range of Ready-To-Eat foods with conventional and real-time RT-PCR for Norovirus GII detection

The influence of measles virus (MV) infection on gene expression by human peripheral blood mononuclear cells (PBMCs) was examined with cDNA microarrays. The mRNA levels of more than 3000 cellular genes were compared between uninfected PBMCs and cells infected with either the Edmonston MV strain or a wild-type MV isolate. The MV-induced upregulation of individual genes identified by microarray analyses was confirmed by RT-PCR. In the present study, a total of 17 genes was found to be upregulated by MV infection. The Edmonston strain grew better in the PBMC cultures than the wild-type MV, and the Edmonston strain was a stronger inducer of the upregulated host cell genes than the wild-type virus. The anti-apoptotic B cell lymphoma 3 (Bcl-3) protein and the transcription factor NF-kappaB p52 subunit were upregulated in infected PBMCs both at the mRNA and at the protein level. Several genes of the interferon system including that for interferon regulatory factor 7 were upregulated by MV. The genes for a number of chaperones, transcription factors and other proteins of the endoplasmic reticulum stress response were also upregulated. These included the gene for the pro-apoptotic and growth arrest-inducing CHOP/GADD153 protein. Thus, the present study demonstrated the activation by MV of cellular mechanisms and pathways that may play a role in the pathogenesis of measles.

Improved detection of Sugarcaneyellowleafvirus using a real-time fluorescent (TaqMan) RT-PCR assay

Concurrent measles infection of cynomolgus monkey during a Reston Ebola virus outbreak in the Philippines

Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens

BackgroundMeasles-Mumps-Rubella (MMR) is an effective live-virus vaccine against measles virus (MeV). However, use of MMR is limited by its inability to boost MeV immunity, lack of immunogenicity in infants, and contraindication in pregnant and immunocompromised persons.MethodsWe evaluated a novel recombinant dimeric MeV hemagglutinin protein vaccine (rMeV) in a rhesus macaque model. Sixteen macaques were primed at day 0 and boosted at day 42 by experimental group: 1) MMR x2; 2) rMeV x2; 3) MMR prime/rMeV boost; 4) control; n=4. Macaques were challenged intratracheally with Bilthoven strain wild type MeV 8 months later. Blood, bone marrow (BM), and lymph node (LN) samples were collected over 3–28 days after challenge. Replication-competent MeV was measured in peripheral blood mononuclear cells (PBMC), BM cells, and LN cells by infectious assay; MeV RNA in PBMC and BM cells was determined by quantitative reverse transcriptase polymerase chain reaction. Plasma was evaluated for MeV-specific IgG and plaque reduction neutralization titer (PRNT).ResultsSix months after vaccination, mean PRNT was 2,432 in rMeV x2 (standard deviation (SD) 3,840), 3,584 in MMR-prime/rMeV boost (SD 3,072) and 5,120 in MMR x2 groups (SD 3,547). Upon infectious challenge, macaques who received any MeV-containing vaccine developed no clinical signs of measles and had no detectable infectious virus in PBMC, BM cells, or LN cells. All unvaccinated macaques had virus in PBMC that peaked at day 7 (mean 3,162 TCID50/mL, SD 4.1) and resolved by day 14 post challenge, and one macaque developed an extensive rash. Macaques who received any MeV-containing vaccine had no detectable MeV RNA in PBMC or BM cells, whereas all unvaccinated macaques had detectable MeV RNA that peaked at day 7 (1.6e5 copies, SD 10.5) in PBMC. In all experimental groups, MeV-specific IgG titers increased after MeV challenge.ConclusionMacaques who received rMeV and/or MMR were protected from rash, viremia, and detection of MeV RNA in PBMC and BM cells, unlike unvaccinated macaques. These data suggest that rMeV vaccine generates protective immune responses against measles and may be a novel candidate for future measles vaccine strategies. Study of cellular responses after rMeV vaccination and MeV challenge is warranted.DisclosuresJessica Rubens, MD, Mevox: Grant/Research Support Guillaume Stewart-Jones, PhD, Moderna: Inventor of SARS-CoV-2 vaccine sequences|Moderna: Stocks/Bonds Michael Watson, MD, MEVOX Ltd: Board Member|MEVOX Ltd: Ownership Interest|MEVOX Ltd: Stocks/Bonds Barney S. Graham, MD, PhD, BSG: BSG is an inventor on patents for the stabilization of the RSV F protein (WO2014160463A1, Prefusion RSV F proteins and their use).|National Institutes of Health: Inventor on patents for RSV vaccines|National Institutes of Health: inventor on patents for measles and other paramyxovirus vaccines Diane Griffin, MD PhD, Gilead: Grant/Research Support|GlaxoSmithKline: Advisor/Consultant|GreenLight Biosciences: Advisor/Consultant|Merck: Advisor/Consultant|MeVox: Grant/Research Support|Takeda Pharmaceuticals: Advisor/Consultant.

IL-5 production by bronchoalveolar lavage and peripheral blood mononuclear cells in asthma and atopy

Engineering Oncolytic Measles Virus to Circumvent the Intracellular Innate Immune Response

BackgroundMorbilliviruses are categorized under the family of Paramyxoviridae and have been associated with severe diseases, such as Peste des petits ruminants, canine distemper and measles with evidence of high morbidity and/or could cause major economic loss in production of livestock animals, such as goats and sheep. Feline morbillivirus (FeMV) is one of the members of Morbilliviruses that has been speculated to cause chronic kidney disease in cats even though a definite relationship is still unclear. To date, FeMV has been detected in several continents, such as Asia (Japan, China, Thailand, Malaysia), Europe (Italy, German, Turkey), Africa (South Africa), and South and North America (Brazil, Unites States). This study aims to develop a TaqMan real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection.ResultsA specific assay was developed, since no amplification was observed in viral strains from the same family of Paramyxoviridae, such as canine distemper virus (CDV), Newcastle disease virus (NDV), and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 × 104 copies/μL with Cq value of 34.32 ± 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34–0.53% and 1.38–2.03%, respectively. In addition, the clinical sample evaluation using this assay showed a higher detection rate, with 25 (35.2%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR.ConclusionsThe TaqMan-based real-time RT-PCR assay targeting the N gene described in this study is more sensitive, specific, rapid, and reproducible compared to the conventional RT-PCR assay targeting the N gene, which could be used to detect early infection in cats.

A real-time TaqMan ® RT-PCR assay with an internal amplification control for rapid detection of transmissible gastroenteritis virus in swine fecal samples

Simultaneous diagnosis of Cetacean morbillivirus infection in dolphins stranded in the Spanish Mediterranean sea in 2011 using a novel Universal Probe Library (UPL) RT-PCR assay

Measles virus (MV) is an enveloped negative strand RNA virus belonging to the family of Paramyxoviridae, genus Morbillivirus, and causes one of the most contagious diseases in humans. Experimentally infected non-human primates are used as animal models for studies of the pathogenesis of human measles. We established a reverse genetics system based on a highly pathogenic wild-type MV. Infection of monkeys with recombinant MV strains generated by reverse genetics enabled analysis of the molecular basis of MV pathogenesis. The essential in vivo function of accessory genes was indicated by infecting monkeys with recombinant MV strains deficient in the expression of accessory genes. Furthermore, recombinant wild-type MV strains expressing enhanced green fluorescent protein enabled visual tracking of MV-infected cells in vitro and in vivo. To date, three different molecules have been identified as receptors for MV. Signaling lymphocyte activation molecule (SLAM, also called CD150), expressed on immune cells, is a major receptor for MV. CD46, ubiquitously expressed in all nucleated cells in humans and monkeys, is a receptor for vaccine and laboratory-adapted strains of MV. The newly identified nectin-4 (also called poliovirus-receptor-like-4) is an epithelial cell receptor for MV. However, recent findings have indicated that CD46 acts as an MV receptor in vitro but not in vivo. The impact of the receptor usage of MV in vivo on the disease outcome is now under investigation.

Introduction: MicroRNAs (miRs) are small non-coding RNAs that regulate the coding RNAs at post-transcriptional level. Some miRs have been shown to act as oncomiRs by promoting growth and invasiveness of tumor cells. One of these oncomiRs, miR-21, has been shown to contribute to tumorigenesis of prostate cancer. Although several effector pathways have been suggested for its oncogenic role, little is known about its upstream regulators. In the present study, we hypothesize that high levels of reactive oxygen species (ROS) produced by the NADPH oxidase regulate the expression of miR-21 and its target proteins, maspin and programmed cell death 4 (PDCD4) in prostate cancer cells and prostate cancer samples. Methods: The levels of miR-21 and NADPH oxidase subunit p47phox were detected in RNA samples from human prostate cancer tissues (from OriGene Technologies) by TaqMan and real time RT-PCR assays, respectively. The levels of maspin and PDCD4 were determined in the formalin-fixed paraffin-embedded (FFPE) prostate tumor sections by immunostaining. In vivo studies were done in severe combined immunodeficient (SCID) mice to study the role of miR-21 and NADPH oxidase in prostate cancer metastasis by injecting transiently-transfected PC3-MM cells with control antisense oligodeoxynucleotides (ODNs), miR-21 antisense ODNs (anti-miR-21), scramble siRNA or p47phox siRNA via the tail vein and counting the metastatic lesions in the lungs. For in vitro studies androgen-independent human prostate cancer cell line PC3-MM was used after transient-transfections with ODNs or siRNAs (as mentioned above). Lysates were used to determine the levels of miR-21 (by TaqMan assay) and maspin, PDCD4 and p47phox (by Western blotting), while invasion and migration was studied by matrigel and wound-healing assays, respectively. Results: RNA samples from patients with different stages of prostate cancer showed high levels of miR-21 and p47phox. This was associated with low expression of maspin and PDCD4, targets proteins which are negatively regulated by miR-21. Studies in SCID mice showed reduced number of metastatic lung lesions in mice injected with anti-miR-21 and p47phox siRNA- transfected PC3-MM cells in comparison to control ODNs and scramble siRNA-transfected cells. Further studies in PC3-MM cells showed reduced expression of miR-21, maspin and PDCD4, and decreased invasiveness and migration of PC3-MM cells in presence of anti-miR-21 and p47phox siRNA. Conclusions: Based on the data from the present study we conclude that NADPH oxidase derived ROS serve as essential regulators of miR-21, its target proteins (maspin and PDCD4), and the oncogenic and pro-invasive functions of miR-21 in prostate cancer cells. The increased expression of p47phox could account for the high levels of ROS generation characteristic of prostate cancer cells and aid in promoting high levels of miR-21 expression and oncogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1184. doi:10.1158/1538-7445.AM2011-1184

The P gene of measles virus (MV) encodes the phosphoprotein, a component of the virus ribonucleoprotein complex, and two nonstructural proteins, C and V, with unknown functions. Growth of recombinant MV, defective in C or V expression, was explored in human peripheral blood mononuclear cells (PBMC). The production of infectious recombinant MV V- was comparable to that of parental MV tag in simian Vero fibroblasts and in PBMC. In contrast, MV C- progeny was strongly reduced in PBMC but not in Vero cells. Consistently, the expression of both hemagglutinin and fusion proteins, as well as that of nucleoprotein mRNA, was lower in MV C--infected PBMC. Thus, efficient replication of MV in natural host cells requires the expression of the nonstructural C protein. The immunosuppression that accompanies MV infection is associated with a decrease in the in vitro lymphoproliferative response to mitogens. MV C- was as potent as MV tag or MV V- in inhibiting the phytohemagglutinin-induced proliferation of PBMC, indicating that neither the C protein nor the V protein is directly involved in this effect.