Optical imaging of nanoscale objects, whether it is based on scattering or fluorescence, is a challenging task due to reduced detection signal-to-noise ratio and contrast at subwavelength dimensions. Here, we report a field-portable fluorescence microscopy platform installed on a smart phone for imaging of individual nanoparticles as well as viruses using a lightweight and compact opto-mechanical attachment to the existing camera module of the cell phone. This hand-held fluorescent imaging device utilizes (i) a compact 450 nm laser diode that creates oblique excitation on the sample plane with an incidence angle of ~75°, (ii) a long-pass thin-film interference filter to reject the scattered excitation light, (iii) an external lens creating 2× optical magnification, and (iv) a translation stage for focus adjustment. We tested the imaging performance of this smart-phone-enabled microscopy platform by detecting isolated 100 nm fluorescent particles as well as individual human cytomegaloviruses that are fluorescently labeled. The size of each detected nano-object on the cell phone platform was validated using scanning electron microscopy images of the same samples. This field-portable fluorescence microscopy attachment to the cell phone, weighing only ~186 g, could be used for specific and sensitive imaging of subwavelength objects including various bacteria and viruses and, therefore, could provide a valuable platform for the practice of nanotechnology in field settings and for conducting viral load measurements and other biomedical tests even in remote and resource-limited environments.

Human herpesvirus 8 (HHV-8

Herpesviruses are characterized as having two distinct life cycle phases: lytic replication and latency. The mechanisms of latency establishment and maintenance, as well as the switch from latency to lytic replication, are poorly understood. Human gammaherpesviruses, including Epstein-Barr virus (EBV) and human herpesvirus-8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), are associated with lymphoproliferative diseases and several human tumors. Unfortunately, the lack of cell lines to support efficient de novo productive infection and restricted host ranges of EBV and HHV-8 make it difficult to explore certain important biological questions. Murine gammaherpesvirus 68 (MHV-68, or ␥HV68) can establish de novo lytic infection in a variety of cell lines and is also able to infect laboratory mice, offering an ideal model with which to study various aspects of gammaherpesvirus infection. Here we describe in vitro studies of the mechanisms of the switch from latency to lytic replication of MHV-68. An MHV-68 gene, rta (replication and transcription activator), encoded primarily by open reading frame 50 (ORF50), is homologous to the rta genes of other gammaherpesviruses, including HHV-8 and EBV. HHV-8 and EBV Rta have been shown to play central roles in viral reactivation from latency. We first studied the kinetics of MHV-68 rta gene transcription during de novo lytic infection. MHV-68 rta was predominantly expressed as a 2-kb immediate-early transcript. Sequence analysis of MHV-68 rta cDNA revealed that an 866-nucleotide intron 5 of ORF50 was removed to create the Rta ORF of 583 amino acids. To test the functions of MHV-68 Rta in reactivation, a plasmid expressing Rta was transfected into a latently infected cell line, S11E, which was established from a B-cell lymphoma in an MHV-68-infected mouse. Rta induced expression of viral early and late genes, lytic replication of viral DNA, and production of infectious viral particles. We conclude that Rta alone is able to disrupt latency, activate viral lytic replication, and drive the lytic cycle to completion. This study indicates that MHV-68 provides a valuable model for investigating regulation of the balance between latency and lytic replication in vitro and in vivo.

The ability to obtain sequence-specific genetic information about rare target organisms directly from complex biological samples at the point of care would transform many areas of biotechnology. Microfluidics technology offers compelling tools for integrating multiple biochemical processes in a single device, but despite significant progress, only limited examples have shown specific, genetic analysis of clinical samples within the context of a fully integrated, portable platform. Herein we present the Magnetic Integrated Microfluidic Electrochemical Detector (MIMED) that integrates sample preparation and electrochemical sensors in a monolithic disposable device to detect RNA-based virus directly from patient samples. By combining immunomagnetic target capture, concentration and purification, reverse-transcriptase polymerase chain reaction (RT-PCR) and single-stranded DNA (ssDNA) generation in the sample preparation chamber, as well as sequence specific electrochemical DNA detection in the electrochemical cell, we demonstrate the detection of influenza H1N1 in throat swab samples at loads as low as 10 TCID50 - 4 orders of magnitude below the clinical titer for this virus. Given the availability of affinity reagents for a broad range of pathogens, our system offers a general approach for multi-target diagnostics at the point-of-care.

Murine gammaherpesvirus 68 (MHV68 [also known as ) is distinguished by its ability to replicate to high titers in cultured cells, making it an excellent candidate for studying gammaherpesvirus virion composition. Extracellular MHV68 virions were isolated, and abundant virion-associated proteins were identified by mass spectrometry. Five nucleocapsid protein homologues, the tegument protein homologue encoded by open reading frame (ORF) 75c, and envelope glycoproteins B and H were detected. In addition, gene products from MHV68 ORF20, ORF24, ORF28, ORF45, ORF48, and ORF52 were identified in association with virions, suggesting that these gammaherpesvirus genes are involved in the early phase of infection or virion assembly and egress.The herpesvirus virion is composed of an icosahedral nucleocapsid surrounded by a proteinacious layer of tegument, which in turn is enclosed by a glycoprotein-containing lipid envelope (50). The structure and protein composition of the nucleocapsid have been shown to be conserved among the three subfamilies (␣Ϫ, ␤Ϫ, and ␥Ϫ) of herpesviruses (11, 14, 62-64, 72, 74). The icosahedral nucleocapsid contains at least four integral structural proteins (the major capsid protein, triplex-1 protein, triplex-2 protein, and small capsid protein) surrounding a core of viral DNA (11,14,27,42,56,62,72,76). The other components of the virion, the envelope and the tegument in particular, are less well understood (38). The envelope contains viral glycoproteins critical for virion binding, entry, and signaling upon infection of a host cell (4,15,26,34,55,67). The tegument is the electron-dense component of the virion surrounding the capsid and interacting with the envelope (14,38,75). While the tegument component of alphaherpesviruses and betaherpesviruses is known to contain a number of gene products involved in assembly and egress of infectious virus (38) or modulation of the host cell environment upon initial infection (10,13,21,25,30,40), little is known about the protein composition of the gammaherpesvirus tegument nor about the functions of gammaherpesvirus tegument proteins immediately after infection of the cell.Study of the functions of tegument proteins in the two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), is hampered by the lack of cell culture systems capable of supporting productive replication of these viruses. However, murine gammaherpesvirus 68 (MHV68, or ␥HV-68) is not constrained in this manner, replicating to high titers in conventional tissue culture systems. MHV68 is a model for studying de novo gammaherpesvirus infection and pathogenesis (16,20,36,66,73). The virus is found in wild murid rodents and is capable of infecting laboratory strains of mice (8,39,48). MHV68 establishes productive infection in lung epithelia and a latent infection in splenocytes, macrophages, dendritic cells, and lung epithelial cells (23,48,57,61,69).The MHV68 virion exhibits morphological similarity to the virion organization of other gammaherpe...

Gammaherpesviruses are known to establish latency in lymphocytes and are associated with tumorigenesis. Two important human pathogens in the family are Kaposi's sarcomaassociated herpesvirus (KSHV; also referred to as HHV-8) and Epstein-Barr virus (EBV). KSHV and EBV are associated with several malignancies, including B-cell lymphomas, nasopharyngeal carcinoma, and Kaposi's sarcoma. Studies of KSHV and EBV are limited by the lack of cell lines to support efficient productive infection and by their restricted host ranges. Murine gammaherpesvirus 68 (MHV-68; also referred to as ␥HV68) is also a member of the gammaherpesvirus family. Unlike KSHV or EBV, in vitro cell culture systems are available to study productive de novo infection by MHV-68, as well as latency and reactivation. MHV-68 forms plaques on monolayers of many cell lines, making it relatively straightforward to genetically manipulate the viral genome. MHV-68 can also establish productive and latent infections in laboratory mice (23), which allows us to pursue questions that relate to host-virus interactions (16,17,20,21). Because of these advantages, MHV-68 offers an excellent model to study the biology and pathogenesis of gammaherpesviruses.Herpesviruses have two distinct phases of their life cycle, productive infection and latency. Reactivation from latency to productive infection is essential for transmission of the virus from host to host and thus is one important aspect of herpesvirus biology. The molecular mechanisms of reactivation have been extensively studied in KSHV and EBV. Cell lines derived from KSHV-or EBV-associated lymphomas are latently infected with virus. A viral protein, Rta (replication and transcription activator) is primarily encoded by open reading frame 50 (ORF50), which is well conserved among gammaherpesviruses. EBV Rta and another viral protein, ZEBRA, function in a cooperative manner to reactivate the viral lytic cycle (2,5,19,27). Although ZEBRA plays a more prominent role in inducing EBV lytic replication (4, 10, 14), Rta alone can disrupt latency in some latently infected cell lines (19,27). KSHV Rta has been shown to be sufficient to reactivate the virus from latently infected B cells derived from KSHV-associated lymphomas (13,22). We have previously shown that MHV-68 Rta is also able to disrupt viral latency and drive viral lytic replication to completion in a latently MHV-68-infected B-cell lymphoma line (26). These studies indicate that Rta of gammaherpesviruses plays a conserved role in virus reactivation.

The herpesvirus life cycle has two distinct phases: latency and lytic replication. The balance between these two phases is critical for viral pathogenesis. It is believed that cellular signals regulate the switch from latency to lytic replication. To systematically evaluate the cellular signals regulating this reactivation process in Kaposi sarcoma–associated herpesvirus, the effects of 26,000 full-length cDNA expression constructs on viral reactivation were individually assessed in primary effusion lymphoma–derived cells that harbor the latent virus. A group of diverse cellular signaling proteins were identified and validated in their effect of inducing viral lytic gene expression from the latent viral genome. The results suggest that multiple cellular signaling pathways can reactivate the virus in a genetically homogeneous cell population. Further analysis revealed that the Raf/MEK/ERK/Ets-1 pathway mediates Ras-induced reactivation. The same pathway also mediates spontaneous reactivation, which sets the first example to our knowledge of a specific cellular pathway being studied in the spontaneous reactivation process. Our study provides a functional genomic approach to systematically identify the cellular signals regulating the herpesvirus life cycle, thus facilitating better understanding of a fundamental issue in virology and identifying novel therapeutic targets.

Gammaherpesviruses establish life-long persistency inside the host and cause various diseases during their persistent infection. However, the systemic interaction between the virus and host in vivo has not been studied in individual hosts continuously, although such information can be crucial to control the persistent infection of the gammaherpesviruses.