Myo1g is required for efficient adhesion and migration of activated B lymphocytes to inguinal lymph nodes.

Cell migration is a dynamic process that involves adhesion molecules and the deformation of the moving cell that depends on cytoskeletal remodeling and actin-modulating proteins such as myosins. In this work, we analyzed the role of the class I Myosin-1 g (Myo1g) in migratory processes of LPS + IL-4 activated B lymphocytes in vivo and in vitro. In vivo, the absence of Myo1g reduced homing of activated B lymphocytes into the inguinal lymph node. Using microchannel chambers and morphology analysis, we found that the lack of Myo1g caused adhesion and chemotaxis defects. Additionally, deficiency in Myo1g causes flaws in adopting a migratory morphology. Our results highlight the importance of Myo1g during B cell migration.

The BeWo cell line derived from a human placental choriocarcinoma is permissive for respiratory syncytial virus infection.

The respiratory syncytial virus (RSV) is the main pathogen associated with upper respiratory tract infections during early childhood. Vertical transmission of this virus has been suggested in humans, based on observations recorded during animal studies that revealed an association of RSV with persistent structural and functional changes in the developing lungs of the offspring. However, human placentas have not yet been evaluated for susceptibility to RSV infection. In this study, we examined the capacity of RSV to infect a human trophoblast model, the BeWo cell line. Our results suggest that BeWo cells are susceptible to RSV infection since they allow RNA viral replication, viral protein translation, leading to the production of infectious RSV particles. In this report, we demonstrate that a human placenta model system, consisting of BeWo cells, is permissive to RSV infection. Thus, the BeWo cell line may represent a useful model for studies that aim to characterize the events of a possible RSV infection at the human maternal-fetal interface.

The Role of Flaviviral Proteins in the Induction of Innate Immunity.

Flaviviruses are positive, single-stranded, enveloped cytoplasmic sense RNA viruses that cause a variety of important diseases worldwide. Among them, Zika virus, West Nile virus, Japanese encephalitis virus, and Dengue virus have the potential to cause severe disease. Extensive studies have been performed to elucidate the structure and replication strategies of flaviviruses, and current studies are aiming to unravel the complex molecular interactions between the virus and host during the very early stages of infection. The outcomes of viral infection and rapid establishment of the antiviral state, depends on viral detection by pathogen recognition receptors and rapid initiation of signalling cascades to induce an effective innate immune response. Extracellular and intracellular pathogen recognition receptors play a crucial role in detecting flavivirus infection and inducing a robust antiviral response. One of the main hallmarks of flaviviral nonstructural proteins is their multiple strategies to antagonise the interferon system. In this chapter, we summarize the molecular characteristics of flaviviral proteins and discuss how viral proteins target different components of the interferon signalling pathway by blocking phosphorylation, enhancing degradation, and downregulating the expression of major components of the Janus kinase/signal transducer and activator of transcription pathway. We also discuss how the interactions of viral proteins with host proteins facilitate viral pathogenesis. Due to the lack of antivirals or prophylactic treatments for many flaviviral infections, it is necessary to fully elucidate how these viruses disrupt cellular processes to influence pathogenesis and disease outcomes.

Generation and characterization of a rat monoclonal antibody against the RNA polymerase protein from Dengue Virus-2.

Dengue virus (DENV) RNA replication requires 2 viral proteins, non-structural protein 3 (NS3) and NS5. NS5 consists of 2 functional domains: a methyltransferase (MTase) domain involved in RNA cap formation and located in the amino terminal region and a RNA-dependent RNA polymerase domain essential for virus replication and located in the carboxyl terminal region. To gain additional insight into the structural interactions between viral proteins and cellular factors involved in DENV RNA replication, we generated a panel of rat monoclonal antibodies (mAbs) against the NS5 MTase domain. Six rat mAbs were selected from 41 clones, of which clone 13G7 was further characterized. The specificity of this antibody for NS5 was demonstrated by western blot of DENV-infected cells, which revealed that this antibody recognizes all 4 DENV serotypes. Furthermore, Western blotting analysis suggested that this antibody recognizes a sequential epitope of the NS5 protein. Positive and specific staining with 13G7 was detected predominantly in nuclei of DENV-infected cells, similarly a pattern was observed in both in human and monkey cells. Furthermore, the NS5 staining co-localized with a Lamin A protein (Pierson index: 0.7). In summary, this monoclonal antibody could be used to identify and evaluate different cellular factors that may interact with NS5 during DENV replication.

Analysis of antibody response in human dengue patients from the Mexican coast using recombinant antigens.

This study was undertaken to evaluate the feasibility of using recombinant dengue proteins to discriminate between acute dengue infections versus uninfected dengue samples. Dengue virus proteins E, NS1, NS3, and NS4B were cloned as fusion proteins and expressed in Escherichia coli. Recombinant products were tested in 100 serum samples obtained from acute dengue fever cases collected from 3 states of Mexico where dengue is endemic. Sera from 75 healthy individuals living in nonendemic areas for dengue were used as a control group. In sera from the dengue patients group, antibody responses to E protein were demonstrated in 91% of cases and NS1 protein was recognized to various extents (99%) within the first 7 days of infection. The antibody responses to NS3 and NS4B were frequently of low magnitude. Consistent negative antibody responses to all proteins were found in sera from the control group. These data suggest that the glutathione-S-transferase (GST)-dengue fusion proteins may be feasible antigens for a sensitive and specific serological assay.

Production and characterization of a monoclonal antibody specific for NS3 protease and the ATPase region of Dengue-2 virus.

Dengue is considered a reemerging disease of worldwide distribution. The Dengue virus non-structural protein 3 (NS3) is known to possess ATPase, helicase, and protease activities that are a constitutive part of the replication complex of Dengue virus. In this report, we discuss the cloning, expressing, and purifying of the Dengue-2 NS3 protein, to immunize mice and then generate monoclonal antibodies (MAbs). Our results show the production of MAbs specific to NS3 protein of Dengue-2 virus, which by immunofluorescence recognize the native protein in experimentally infected endothelial cells (HMEC). Likewise, C6/36-infected lisates were used in Western blots, and observed the specific characteristic band that defines the NS3 protein. We conclude that these antibodies may be a useful tool, not only to study the replicative process of Dengue virus, but also to generate specific diagnostic tools for Dengue infection.