Laboratory of Molecular Virology

Bethesda, MD, United States

Laboratory of Molecular Virology

Bethesda, MD, United States
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Bergallo M.,University of Turin | Montanari P.,University of Turin | Mareschi K.,Aou Citta Della Salute E Della Science Regina Margherita Children Hospital | Rassu M.,University of Turin | And 2 more authors.
Journal of Virological Methods | Year: 2017

TaqMAMA is an allele-specific PCR-based (ASPCR) method that may be suitable for broad and cost-effective genotyping applications in all types of laboratories. There is evidence that interactions between some toll like receptors (TLRs) with viruses influence both the immune response and outcome of HCMV infection. We developed a TaqMAMA genotyping assay for the detection of rs352140 TLR9 polymorphism in transplant recipients with and without HCMV infections. Performance parameters to ensure a solid pre-validation protocol have been here argued. We analysed a population of 74 kidney transplants recipients subdivided in 58 HCMV PCR positive and 16 HCMV PCR negative in the post-transplant routine control. All 74 samples were tested with 31/74 (41.9%) homozygotes (11 CC and 20 TT) and 43/74 (58.1%) heterozygotes (CT). Our preliminary data suggest that there is no correlation between TLR9 rs352140 polymorphism and frequency of HCMV infection. © 2017 Elsevier B.V.

Wang X.,Laboratory of Molecular Virology | Tan J.,Laboratory of Molecular Virology | Zoueva O.,U.S. Food and Drug Administration | Zhao J.,Laboratory of Molecular Virology | And 2 more authors.
Microbes and Infection | Year: 2014

It is not well-known whether apoptosis signaling affects influenza virus infection and reproduction in human lung epithelial cells. Using A549 cell line, we studied the relationship of some apoptosis-associated molecules with novel pandemic influenza A (H1N1) virus, A/California/04/2009. Infected cells displayed upregulated Fas ligand, activated FADD and caspase-8, and downregulated FLIP in the extrinsic apoptotic pathway. p53 expression increased and Bcl-XL expression decreased in the intrinsic pathway. Expression of pre-apoptotic molecules (FasL, FADD, and p53) increased virus replication, while inhibition of activity of FADD, caspase-8 and caspase-3, and expression of anti-apoptotic proteins (FLIP and Bcl-XL) decreased virus replication. p38, ERK and JNK from MAPK pathways were activated in infected cells, and inhibition with their inhibitors diminished virus replication. In the p38 superfamily, p38α expression increased viral RNA production, while expression of p38β and p38γ decreased. These data indicated that influenza virus induces apoptotic signaling pathways, which benefit virus replication. © 2013.

Wang X.,Laboratory of Molecular Virology | Tan J.,Laboratory of Molecular Virology | Zhao J.,Laboratory of Molecular Virology | Ye Z.,U.S. Food and Drug Administration | Hewlett I.,Laboratory of Molecular Virology
BMC Infectious Diseases | Year: 2014

Background: Highly pathogenic avian influenza A virus has been shown to infect organs other than the lung, and this is likely to be mediated by systemic spread resulting from viremia which has been detected in blood in severe cases of infection with avian H5N1 viruses. The infectivity of virus in blood and the potential for virus transmission by transfusion has not been investigated.Methods: Using a susceptible ferret animal model, we evaluated viremia and transmission by blood transfusion. Blood was collected on day 2, 4, 6, and 10 post-infection (or before death) from donor ferrets infected with either low dose (1.0 × 102.6 EID50/ml) or high dose (1.0 × 103.6 EID50/ml) of H5N1 virus, A/VN/1203/04 and transfused to recipient animals.Results: Viremia was observed in 2/12 (16.67%) recipients that received blood from donor ferrets infected with low dose and 7/12 (58.33%) recipients who received blood from high dose infected donors. 1/12 (8.3%) low dose recipients and 6/12 (50%) high dose recipients died within 11 days after transfusion. Increased changes in body weight and temperatures were observed in high dose recipients, and high levels of viral RNA were detected in recipient ferrets after transfusion of blood from the early viremic phase, which also correlated with adverse impact on their survival.Conclusion: These data suggest that highly pathogenic avian influenza A virus, H5N1, is transmissible by blood transfusion in ferrets. Low levels of viremia were detected around the time of onset of symptoms and later in ferrets infected with highly pathogenic H5N1 virus. These findings may have implication for pathogenesis and transmissibility of H5N1. © 2014 Wang et al.; licensee BioMed Central Ltd.

Wang X.,Laboratory of Molecular Virology | Zoueva O.,U.S. Food and Drug Administration | Zhao J.,Laboratory of Molecular Virology | Ye Z.,U.S. Food and Drug Administration | Hewlett I.,Laboratory of Molecular Virology
BMC Infectious Diseases | Year: 2011

Background: Influenza A virus has been detected in the blood of some infected individuals, and may pose a safety concern for collection, handling and transport of specimens for epidemiological and public health investigations if infectious virus is present in samples. Furthermore the effect of storage on virus stability and infectivity has not been well studied.Methods: We examined the stability of novel pandemic influenza A (H1N1) virus RNA when the virus was stored in phosphate buffered saline (PBS), plasma, or buffy coated blood at either room temperature or 4°C using a sensitive Taqman RT-PCR assay. We also investigated virus infectivity using the EID 50assay when virus was stored in PBS, plasma, or buffy coats isolated from blood at 4°C.Results: Viral RNA stability was affected by the matrix used for storage. The recovery of viral RNA was highest when virus was stored in PBS with lower amounts being recovered from plasma and buffy coats at either room temperature or 4°C. Incubation time did not appear to be a major factor for viral RNA stability, although there was gradual decline after longer periods post-incubation. Both sample matrix and incubation time affected virus infectivity. The decay in virus infectivity was greatest in PBS followed by buffy coats and plasma. Virus infectivity was abolished in buffy coats at day 20 post-incubation when virus concentrations were low.Conclusion: These data indicate that encapsidated viral RNA was stable overall in all three liquid matrices at room temperature or 4°C although it was most stable in PBS; virus infectivity in buffy coats at 4°C decayed in a time dependent manner while it remained unchanged in plasma. These findings have implications for storage, handling and transport of blood derived samples from influenza patients for epidemiological and laboratory investigations. It should be noted that there is little known about influenza viremia, and whether influenza viruses can be transmitted by blood or blood derived samples. © 2011 Wang et al; licensee BioMed Central Ltd.

PubMed | U.S. Food and Drug Administration and Laboratory of Molecular Virology
Type: Journal Article | Journal: Viruses | Year: 2016

Influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. It is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (HIV)-1 replication in HIV-1-infected patients. Using a lymphoma cell line, Jurkat, we examined the in vitro effects of pandemic influenza A (H1N1) virus (pH1N1) infection on cell death and HIV-1 RNA production in infected cells. We found that pH1N1 infection increased apoptotic cell death through Fas and Bax-mediated pathways in HIV-1-infected Jurkat cells. Infection with pH1N1 virus could promote HIV-1 RNA production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B), nuclear factor of activated T-cells (NFAT) and activator protein 1 (AP-1) through mitogen-activated protein kinases (MAPK) pathways and T-cell antigen receptor (TCR)-related pathways. The replication of HIV-1 latent infection could be reactivated by pH1N1 infection through TCR and apoptotic pathways. These data indicate that HIV-1 replication can be activated by pH1N1 virus in HIV-1-infected cells resulting in induction of cell death through apoptotic pathways.

Farci D.,University of Cagliari | Bowler M.W.,European Molecular Biology Laboratory | Bowler M.W.,French National Center for Scientific Research | Kirkpatrick J.,European Molecular Biology Laboratory | And 3 more authors.
Biochimica et Biophysica Acta - Biomembranes | Year: 2014

We have analyzed the cell wall of the radio-resistant bacterium Deinococcus radiodurans. Unexpectedly, the bacterial envelope appears to be organized in different complexes of high molecular weight. Each complex is composed of several proteins, most of which are coded by genes of unknown function and the majority are constituents of the inner/outer membrane system. One of the most abundant complexes is constituted by the gene DR-0774. This protein is a type of secretin which is a known subunit of the homo-oligomeric channel that represents the main bulk of the type IV piliation family. Finally, a minor component of the pink envelope consists of several inner-membrane proteins. The implications of these findings are discussed. © 2014 Elsevier B.V.

Bergallo M.,University of Turin | Galliano I.,University of Turin | Montanari P.,University of Turin | Gambarino S.,University of Turin | And 6 more authors.
Journal of Clinical Virology | Year: 2015

Objectives: The aim of this study was to evaluate HERV-K and -W pol gene expression in kidney transplant recipients and to investigate the possible relationship between HERVs gene expression and CMV infection in these patients. Study design: Thirty-three samples of kidney transplant patients and twenty healthy blood donors were used to analyze, HERV-K and -W pol gene RNA expression by relative quantitative relative Real-Time PCR. Result: We demonstrated that HERVs pol gene expression levels were higher in kidney transplant recipients than in healthy subjects. Moreover, HERV-K and -W pol gene expression was significantly higher in the group of kidney transplant recipients with high CMV viral load than in the groups with no or moderate CMV viral load. Conclusion: Our data suggest that CMV may facilitate in vivo HERV activation. © 2015.

PubMed | Federal University of Minas Gerais, Secretary for Health, Laboratory of Molecular Virology, Federal University of Santa Catarina and 2 more.
Type: | Journal: Scientific reports | Year: 2016

Targeting regions of proteins that show a high degree of structural conservation has been proposed as a method of developing immunotherapies and vaccines that may bypass the wide genetic variability of RNA viruses. Despite several attempts, a vaccine that protects evenly against the four circulating Dengue virus (DV) serotypes remains elusive. To find critical conserved amino acids in dengue viruses, 120 complete genomes of each serotype were selected at random and used to calculate conservation scores for nucleotide and amino acid sequences. The identified peptide sequences were analysed for their structural conservation and localisation using crystallographic data. The longest, surface exposed, highly conserved peptide of Envelope protein was found to correspond to amino acid residues 250 to 270. Mutation of this peptide in DV1 was lethal, since no replication of the mutant virus was detected in human cells. Antibodies against this peptide were detected in DV naturally infected patients indicating its potential antigenicity. Hence, this study has identified a highly conserved, critical peptide in DV that is a target of antibodies in infected humans.

Ravanini P.,Laboratory of Molecular Virology | Huhtamo E.,University of Helsinki | Ilaria V.,Laboratory of Molecular Virology | Crobu M.G.,Laboratory of Molecular Virology | And 9 more authors.
Eurosurveillance | Year: 2012

Mosquitoes collected in northern Italy were screened for flavivirus RNA. Positive amplicons were sequenced and found most similar to insect flavivirus (ISF), Usutu virus (USUV) and surprisingly also to Japanese encephalitis virus (JEV). The sequence (167 bp), obtained from one pool of Culex pipiens, was found identical to JEV strains from bats in China. Unfortunately additional sequence data or virus isolations were not obtained in this study. Confirmation of potential introduction of JEV to Italy and other European countries is urgently needed.

PubMed | Laboratory of Molecular Virology
Type: Journal Article | Journal: AIDS research and human retroviruses | Year: 2016

Accurate detection and quantification of HIV-1 group O viruses have been challenging for currently available HIV assays. We have developed a novel time-resolved fluorescence (TRF) europium nanoparticle immunoassay for HIV-1 group O detection using a conventional microplate enzyme-linked immunosorbent assay (ELISA) and a microchip platform. We screened several antibodies for optimal reactivity with several HIV-1 group O strains and identified antibodies that can detect all the strains of HIV-1 group O that were available for testing. The antibodies were used to develop a conventional ELISA format assay and an in-house developed europium nanoparticle-based assay for sensitivity. The method was evaluated on both microwell plate and microchip platforms. We identified two specific and sensitive antibodies among the six we screened. The antibodies, C65691 and ANT-152, were able to quantify 15 and detect all 17 group O viruses, respectively, as they were broadly cross-reactive with all HIV-1 group O strains and yielded better signals compared with other antibodies. We have developed a sensitive assay that reflects the actual viral load in group O samples by using an appropriate combination of p24 antibodies that enhance group O detection and a highly sensitive TRF-based europium nanoparticle for detection. The combination of ANT-152 and C65690M in the ratio 3:1 was able to give significantly higher signals in our europium-based assay compared with using any single antibody.

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