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Rapid and sensitive point-of-care diagnostics are of paramount importance for early detection of infectious diseases and timely initiation of treatment. Here, we present cellulose paper and flexible plastic chips with printed graphene-modified silver electrodes as universal point-of-care diagnostic tools for the rapid and sensitive detection of microbial pathogens or nucleic acids through utilizing electrical sensing modality and loop-mediated isothermal amplification (LAMP). We evaluated the ability of the developed paper-based assay to detect (i) viruses on cellulose-based paper microchips without implementing amplification in samples with viral loads between 106 and 108 copies per ml, and (ii) amplified HIV-1 nucleic acids in samples with viral loads between 10 fg µl−1 and 108 fg µl−1. The target HIV-1 nucleic acid was amplified using the RT-LAMP technique and detected through the electrical sensing of LAMP amplicons for a broad range of RNA concentrations between 10 fg µl−1 and 108 fg µl−1 after 40 min of amplification time. Our assay may be used for antiretroviral therapy monitoring where it meets the sensitivity requirement of the World Health Organization guidelines. Such a paper microchip assay without the amplification step may also be considered as a simple and inexpensive approach for acute HIV detection where maximum viral replication occurs.

During the past decade or so, there has been a substantial body of work to dissect arboviral evolution and to develop models of adaptation during host switching. Regardless of what species serve as host or vectors, and of the geographic distribution and the mechanisms of replication, arboviruses tend to have slow evolutionary rates in nature. The hypothesis that this is the result of replication in the disparate environments provided by host and vector did not receive solid experimental support in any of the many viral species tested. Instead, it seems that from the virus’s point of view, either the two environments are sufficiently similar or one of the environments so dominates viral evolution that there is tolerance for suboptimal adaptation to the other environment. Replication in alternating environments has an unexpected cost in that there is decreased genetic variance that translates into a compromised adaptability for bypassed environments. Arboviruses under strong and continuous positive selection may have unusual patterns of genomic changes, with few or no mutations accumulated in the consensus sequence or with dN/dS values typically consistent with random drift in DNA-based organisms.

Background/Aims - In Epstein-Barr virus (EBV) positive cell lines that are stably infected, three different promoters are known to direct the transcription of EBV nuclear antigen 1 (EBNA1). These are located in the BamHI-C, BamHI-Q, and BamHI-F regions of the viral genome (Cp, Qp, and Fp, respectively). Fp is activated upon induction of the viral lytic cycle. The aim of this study was to investigate the activity of Fp in EBV associated diseases. Methods - Using reverse transcriptase polymerase chain reaction, a qualitative analysis of EBNA1 promoter usage in various EBV associated diseases was performed. Results - Fp driven transcription was detected in the context of primary infection and/or lytic replication; at least a portion of the Fp driven transcripts encoded EBNA1. Qp driven EBNA1 transcripts were detected in most samples across the range of disorders tested. Cp driven EBNA1 transcripts were detected in the context of immune suppression and in samples containing EBV positive (nonneoplastic) lymphoid cells. Conclusions - These results confirm the previously proposed housekeeping function of the Qp promoter., published_or_final_version

To assess dried plasma spots (DPSs) as a source of material for virus quantification, human immunodeficiency virus type 1 (HIV-1) RNA levels were quantified in matched DPS and liquid plasma samples from 73 infected patients, including 5 neonates and 4 adult patients with acute HIV-1 infection. Quantifications were performed by commercially available assays (NASBA [nucleic acid sequence-based amplification] or Amplicor, or both). There was a strong correlation between HIV-1 RNA levels in plasma and DPSs. More importantly, there was no decline in HIV-1 RNA levels in DPSs stored for as long as 2 weeks at 20 degrees C. Similarly, storage of DPSs for 3 days at 37 degrees C resulted in no decrease in viral RNA levels. For patients with primary infection, the DPS method allowed for the measurement of RNA levels in plasma during the initial spike in the level of viremia and in the subsequent period of suppressed viral replication. DPS quantification was equally informative in the neonatal setting, with all five newborns showing HIV-1 RNA loads of greater than 4.991 log10 copies/ml. We conclude that the viral RNA levels in DPSs are equivalent to those measured in fresh-frozen plasma. The ease and economy of DPS sampling, the minute volumes required, and the unexpected stability of dried RNA suggest that the use of DPSs will be particularly valuable for small-volume neonatal samples and large, population-based studies in which cold storage and transportation present special problems, as is often the case in developing countries. The ability to measure viral changes during primary infection suggests that the method will be useful for assessing vaccine efficacy in large field trials.