The combination of viral immuno-specificity due to surface functionalization and the physical size measurements enabled by holographic imaging is used to sensitively detect and enumerate HSV particles using our compact and cost-effective platform. This computational sensing technique can find numerous uses in global health related applications in resource-limited environments.Caused by the herpes simplex virus (HSV), herpes is a viral infection that is one of the most widespread diseases worldwide. Here we present a computational sensing technique for specific detection of HSV using both viral immuno-specificity and the physical size range of the viruses. This label-free approach involves a compact and cost-effective holographic on-chip microscope and a surface-functionalized glass substrate prepared to specifically capture the target viruses. To enhance the optical signatures of individual viruses and increase their signal-to-noise ratio, self-assembled polyethylene glycol based nanolenses are rapidly formed around each virus particle captured on the substrate using a portable interface. Holographic shadows of specifically captured viruses that are surrounded by these self-assembled nanolenses are then reconstructed, and the phase image is used for automated quantification of the size of each particle within our large field-of-view, ~30 mm Once contracted, HSV establishes a lifetime latent infection, with periodic outbreaks that lead to painful sores and blisters around the mouth, eye and genitals. Some of the additional complications also include fever and sore throat among others. Recent studies have shed light onto the viral structure of HSV and its interaction with the host cells. The viral DNA is packed inside the capsid, which is surrounded by a layer of proteins called the tegument that is further enveloped in the lipid bilayer. The virus in its dormant state is generally located in the axons of peripheral nervous system neurons and can travel to epithelial cells leading to an outbreak5. However, the virus can still be transmitted if no active symptoms are present. Additionally it has been well documented that people carrying this virus may not show any symptoms for a long period of time and be unaware of this infection. These factors significantly increase the likelihood of spreading this virus unknowingly. It has been estimated that the total sexually-transmitted-diseases (STD) related medical costs in the US over the last decade was over 15 billion USD, with HSV alone costing more than 500 million USD6. This obviously outlines the need for proper and timely screening of HSV to prevent its wide-spread transmission and ensure proper treatment. Patient management studies have clearly shown the benefits of early testing, as it helps in definitive diagnosis, which leads to proper course of therapy as well as counseling for the patients and their significant others7.Herpes is one of the most common viral diseases, which is caused by a DNA based virus, i.e., the herpes simplex virus (HSV). There are two different types of HSV: HSV-1, responsible for oral herpes and HSV-2, which causes genital herpes. It is estimated that >50% of Americans between 14–49 years of age have tested seropositive for this virus8. PCR is an extremely sensitive technique that can be used to detect viruses with great sensitivity and specificity9. In addition to PCR, loop mediated isothermal amplification (LAMP) based methods have also been used for the detection of HSV10. Serological tests that sense HSV specific antibodies using techniques such as enzyme-linked immunosorbent assays (ELISA)12, western blot13, antibody-dependent cellular lysis, and radio-immunoassays are also available. Some other HSV detection methods include immunofluorescence microscopy14, luciferase immunoprecipitation assay15, Tzanck smear16 and electron microscopy17. Some of these immunoassay based tests, however, can have significant amounts of incorrect results19. It was previously reported that the sensitivity and specificity of enzyme based immunoassay techniques compared to culture was 65% and 93%, respectively20. Compared to culture, the specificity was close to 100% and the sensitivity varied between 50% and 100% for direct fluorescence immunoassay based approaches21. Among the currently used clinical methods, PCR has been shown to have quite high sensitivity and specificity. For example, the commercially available PCR based MultiCode-RTx kit  has a reported sensitivity and specificity of 92.4% and 98.3%, respectively for HSV-1 and 95.2% and 93.6%, respectively for HSV-221. PCR based techniques also have a very good limit of detection, typically ~10 viral particles per assay22. However, PCR, along with most of the other techniques, require expert handling and relatively costly and bulky instrumentation, which limit wide-scale availability of these tests, particularly in rural areas and resource limited settings.Currently the two most commonly used clinical HSV diagnostic techniques are viral culture and polymerase chain reaction (PCR). Viral culture involves extraction of the virus from the lesions, using swabs and sterile needles, and culturing them for several days, followed by different assays such as hemagglutination30 and in this work we utilize this computational imaging framework for specific and sensitive detection and enumeration of HSV-1 particles. In our holographic microscope, the substrate is illuminated from the bottom using a programmable-array of LEDs and the interference pattern  formed due to the scattered optical wave from the viruses and the non-diffracted wave, is recorded on a CMOS imager chip placed close to the sample, with sub-mm gap between the viral particles and the sensor active area. The optical signal from each virus particle is further boosted by using nanolenses, which are self-assembled around the viruses using polyethylene glycol (PEG) vapor. A unique feature of this lensfree imaging platform is that both amplitude and phase information of the objects are digitally reconstructed from the holographic diffraction patterns captured by the image sensor chip. In this computational microscope, a set of e.g., 20 sub-pixel shifted images is captured and then a super-resolved hologram is obtained by using a lateral-shift based pixel super-resolution method33. Following the reconstruction of the super-resolved hologram, quantitative phase information of the captured particles is also obtained. Based on the information of this phase channel, the size of the particles can be determined using a calibration curve that is established with particles of known size . The sizing accuracy of this approach was determined to be ±11 nm29. Similarly, we used this phase information to estimate the size of the virus, which acts as a second level of screening in addition to surface chemistry, eliminating the need for any fluorescent labeling. This mobile virus imaging and sensing platform, weighing <500 g, can not only determine the presence or absence of the virus within the sample but can also estimate the viral count of the sample.Here we present a cost-effective and easy to use mobile platform for HSV-1 detection, using a portable lensfree holographic microscope with pixel super-resolution capabilities. In this label-free sensor design, HSV-1 viral particles, which are ~150–200 nm in size, are detected using a compact holographic microscope after capturing them on specially prepared substrates, using virus-specific antibodies. This approach allows us to utilize antibodies to specifically capture the virus on a substrate and use the computational imaging based microscopic sizing as a secondary method of validation and detection, thus eliminating the necessity for labeling with fluorophores or enzymes. Recently, lensfree holographic imaging has shown promising results for imaging and detection of nanoparticles due to its wide field-of-view (FOV), cost effectiveness and simplicity35). This is done by first capturing the target viral particles onto a glass substrate using virus-specific antibodies, as shown in Fig. The schematic of the lensfree on-chip holographic microscope is shown in Fig. To test the capture efficacy of the substrate and the nonspecific binding rate, we performed control experiments using biotin tagged 0.25 µm beads and uncoated 1 µm fluorescent beads, as shown in Fig. 37. Note that there are several glycoproteins on the envelop of each HSV particle34 that can bind to multiple antibodies, which enable the virus to be labeled with fluorescent molecules and biotin at the same time using multiple antibodies. We conveniently used this approach to cross-validate the efficacy of our surface chemistry technique.Next, we tested the specificity of the substrate using HSV-1 particles. This is done in three ways: by using (i) antibody conjugated HSV-1 on unmodified substrates, (ii) HSV-1, without any antibody conjugation, on fully prepared substrates, and (iii) antibody conjugated HSV-1 on fully prepared substrates. When antibody conjugated HSV-1 particles are added to an unmodified glass substrate, without any PEG coverage, we observe a significant amount of nonspecific binding of the virus. The viral particles cover the full surface area of the substrate due to non-specific binding see Fig. . We next35 and we used this information to digitally reject non-specifically bound particles that are outside of this range as ‘non-viral’, which helps to increase our specificity. Nanoscopic size measurements are typically performed using electron microscopy (which is very accurate but tedious to use and expensive) or dynamic light scattering based approaches (which are limited in accuracy as well as the dynamic range of the particle size and density that can be measured). Our lensfree computational microscopy platform provides a compact and cost-effective method of not only imaging viruses, as shown in Fig. 29. This range of concentration is clinically relevant for both HSV1 and HSV2, which is on the order of ~102–1010 DNA copies per ml depending on the sample and collection method38.In addition to surface functionalization and immuno-specific capture of the viruses, the specificity of our mobile platform is further improved by digitally incorporating the physical size of the viral particles into the automated decision process. The HSV particle size is approximately 150–200 nm in diameter29. We also imaged the same particles using SEM  by manually selecting a region of interest and then implemented an automated size based counting algorithm on the selected region. In order to illustrate the sizing of the captured virus particles we randomly selected three particles as shown in Fig. see Fig.  inset an2) by imaging multiple regions of interest within a given substrate - see Fig. 2 of our FOV  may be due to non-specific nanolens formation on the substrate and/or due to error in our HSV-1 detection, sizing and counting algorithms. Note that if an imager chip with a smaller active area is used, the total volume of the assay will need to be reduced, proportional to the area of the imager chip (assuming that the sample volume per mm2 of our FOV remains the same). Therefore, for different on-chip microscope designs that employ other CMOS imagers, the assay volume per test can be accordingly adjusted by simple centrifugation of the sample. Significantly increasing the test sample volume per unit area of the chip would also increase false positives as well as spatial overlap related misses, especially at higher virus concentrations.In order to estimate the limit of detection (LOD) of our computational HSV sensor, we measured various samples, each containing a different number of virus particles, with one acting as a control without any viruses. In these experiments, virus particles were suspended within a volume of 50–100 µL per test and we counted HSV-1 particles per unit area , which could enable multiplexed testing for different viruses in parallel, by e.g., spotting antibodies for a panel of viruses on the same disposable substrate.One should note that our experiments were performed using HSV-1 particles from culture. In a clinical setting, the virus is extracted from lesions using swabs and then suspended in a buffer, which can then be used for imaging/sensing using the presented computational microscope. The viral samples can also be concentrated by centrifugation and re-suspended in appropriate amount of buffer to be adjusted per test . This computational method of viral particle sensing can be extended to various other types of viruses as well. Counting the number of viral particles in a given sample can not only help determine the severity of the disease but also monitor the efficacy of an anti-viral therapy. One of the important advantages of the presented imaging system is its ability to image over a wide field-of-view . The viral culture  was acquired from ZeptoMetrix Corporation. All the chemicals and materials were used as received.39. The antibodies were used at a dilution of 1:20 as specified by the company. The same step can be similarly performed in serum. The solution was then centrifuged to remove any unreacted antibody and re-suspended in water. The antibody conjugated virus was then added to the streptavidin coated coverslip as a droplet, and incubated for more than an hour for the biotin to bind with streptavidin. The sample is then washed thoroughly with water following this incubation step. The droplet is approximately the size of the sensor area. The volume of the viral solution per test was varied from ~50 µl to a maximum of 100 µl, depending on the concentration of the initial solution. Varying the test volume in this small range does not significantly affect the number of false positives in the size range of HSV (~150–200 nm) , placed inside the reservoir. The temperature is set and monitored using a computer-controlled feedback temperature controller, with a thermistor  immersed in the PEG solution. The temperature control unit is interfaced to the main PC through a LabVIEW program as well.The experiments are performed by first acquiring a set of images before the PEG condensation, which serves as a baseline to the evaporation process. The temperature of the PEG is then increased to ~95 °C, which results in evaporation and subsequent PEG deposition/condensation on the sample and formation of the nanolenses. The lensfree holographic images are continuously acquired during this process to monitor the evolution of the nanolenses and to determine the optimal time point for virus detection. Image reconstruction and analysis using our GUI takes only a few minutes. Following the completion of the imaging process, at the sample we observe a small temperature change of about 4 °C, from 27 °C to 31 °C , while eliminating other regions. First, we used an edge detection algorithm and outlined the edges of the holograms. Then we performed Hough transformation to spot out the circularly shaped holograms. These selected areas are used for virus detection and counting (per unit area) and the rest of the FOV is discarded.29, where the holographic image reconstruction and all the digital processing steps are performed using a custom developed MATLAB program running on a PC . After inputting the correct holographic reconstruction parameters, including the wavelength and the effective pixel size, the algorithm first reconstructs pixel super-resolved images of the sample at different sample-to-sensor distances (z2). Using this stack of z2 images, some spurious particle-like noise features are eliminated by incorporating a focusing criterion for particle identification. Stated differently, real particles show a gradual change in their phase signal as a function of z2, whereas particle-like noise terms do not exhibit a similar focusing behavior. We used this physical focusing criterion to remove spurious particle-like features, enabling proper identification and counting of the specifically-captured viral particles. In this digital counting process, the larger particles, which have higher peak phase values in our reconstructed phase images, are counted first. Then, they are digitally removed along with their twin images, which often mask the smaller particles located in close proximity to larger particles29. Such larger particles are digitally removed by replacing their amplitude and phase information with the average of the background in the twin-image plane. The above described steps are repeated multiple times till all the particles are automatically counted.The automated viral particle counting is performed using a segmentation algorithmA bright-field and fluorescence microscope  was used for comparison purposes. These comparison images were acquired with a scientific charge-coupled device (CCD) camera  by optimizing the gain and exposure time of the camera for each modality. Fluorescence imaging of the viral particles was performed by first tagging the virus with FITC conjugated antibodies. These HSV-1 particles were imaged using a 40× objective lens and the sample was irradiated with 488 nm excitation light and the fluorescence light was observed at ~532 nm.The specific binding studies were performed using two different fluorescent beads. 0.25 µm green fluorescent beads with biotin coating and 1 micron red fluorescent beads were mixed in equal amounts and deposited on the substrate. After an hour of incubation the beads were thoroughly washed using water and imaged under the fluorescence microscope. The green fluorescent beads were imaged using the 488 nm excitation filter and 532 nm emission filter, whereas the red beads were imaged using the 532 nm excitation filter and the 632 nm emission filter.To provide image comparisons, some of the virus samples under test were first coated with gold using a sputter coater (Denton Desk V) with a thickness of around 10 nm. The samples were then imaged using an SEM (Nova 600 SEM/FIB System). The following steps were performed to register the images, which is used for comparison purposes. First, easily identifiable marks were placed on the sample slides, before imaging with the lensfree platform. These marks are clearly visible in the reconstructed images, and during SEM imaging , serve as the main anchors for image registration. Prior to SEM imaging, secondary anchor points (larger particles that are easier to register) are identified near the target particles. The horizontal and vertical distances of these targets with respect to the secondary anchors are calculated from the reconstructed image to form a roadmap. During the SEM imaging, the sample is first aligned such that the angular orientation of the slide matches the reconstructed image. Once the secondary anchors are identified, each target particle is visited by shifting the sample holder by appropriate distances. This step-by-step particle searching strategy enables us to register our images accurately, which is used for comparison.Supplementary Information


An infectious agent new to Brazil arrived in our country in 2015, to cause significantsocial impact and uproar in the scientific community in the areas of human health andreproduction. The Zika virus, detected in past decades in Africa and Southeast Asia,first appeared in the Brazilian Northeast in October of the said year, primarily in theState of Pernambuco, where 25.9% of the suspected cases were observed. The outbreakstretched into the following year, causing civil unrest, the issuance of health alerts,and apprehension in the scientific community as more information and studies on theinfectious disease were needed . But the Zikavirus is not transmitted solely through a simple mosquito bite. Other pathways includeblood transfusion, sexual intercourse, and transplacental transmission. Additionalpossibilities are being studied -PCR asmany as 188 days after infection (Despite the lack of studies devised to gather more evidence on the presence and behaviorof the virus in gametes and in the reproductive apparatus of individuals of both sexes,semen infected with the Zika virus has been the topic of several study submissionslooking into pathogen-gamete interactions and the possibility of infectious materialsmaking it to human oocytes. In published studies, gametes and animal embryos weresusceptible to infection by viral antigens despite glycoprotein-based protectionmechanisms such as the zona pellucida, although long-term developmental implicationshave not been analyzed. These findings may serve as the foundation for the risk factorsconnected to the transmission of Zika virus in assisted reproductive technologies, sincefollicular fluid may carry the Zika virus (Although the site of viral replication in humans has not been determined, it is believedto be located in the prostate or seminal vesicles, preferentially in the testicular andepididymal region, potentially extending the duration of the sexual transmissionpotential. Through analogies with other studies on brain tissue, the Zika virus isbelieved to preferentially infect progenitor cells, making spermatogonia its maintarget. The appearance of orchitis and epididymitis in mice up to sixty days afterinfection demonstrated the potential of the pathogen to cause tissue damage in thelong-term by innate immune response. Evidence of interference with cell division in thetesticles poses another significant challenge to male fertility, since it may induce thedeath of spermatozoa, infertility, and potentially azoospermia, in addition to possibleeffects on mature sperm cells such as decreasing their motility and ability to fertilizeoocytes. The female genitourinary tract has also been analyzed as a possible reservoirof ZIKV and a potential source of chronic risk to fetuses, two factors thatsignificantly complicate prenatal counseling. Nonetheless, the role of bodily fluids andthe urinary systems of both sexes in the extension of the infectious potential of viralRNA needs to be better investigated and clarified (Fear of the complications associated with infection by the Zika virus have led to areduction in the number of births in the State of Pernambuco. Hospitals once overcrowdedstarted to have empty beds by the end of 2016. In September of 2015, before the issuanceof Zika virus warnings, some 8,000 births were recorded in the State of Pernambuco. InNovember of 2016, just over ten months since the declaration of state of emergency onaccount of cases of microcephaly, the number of births dropped by an astounding 27%. InCampinas, São Paulo, the number of births decreased by more than 5.0% in 2016 inrelation to 2015, breaking a decade-long string of annual growth rates of two percent.Two reasons were listed by the local Secretary of Health to explain the decrease in thenumber of births: the economic crisis the country is going through, which may haveforced couples to postpone their plans of having children, and the cases of Zika virusinfection, which significantly increased the number of babies born with microcephaly. InBelo Horizonte, Minas Gerais, many maternity hospitals recorded decreases in the numberof births in the first trimester of 2017. The State's Capital also had cases of Zikavirus infection. The combination of generalized fear of an outbreak and awareness of thedisease's complications pulled the number of births down by more than 20%, particularlyamong women aged 20-30 years.


Influenza virus intimately associates with host RNA polymerase II (Pol II) and mRNA processing machinery. Here, we use mammalian native elongating transcript sequencing (mNET-seq) to examine Pol II behavior during viral infection. We show that influenza virus executes a two-pronged attack on host transcription. First, viral infection causes decreased Pol II gene occupancy downstream of transcription start sites. Second, virus-induced cellular stress leads to a catastrophic failure of Pol II termination at poly(A) sites, with transcription often continuing for tens of kilobases. Defective Pol II termination occurs independently of the ability of the viral NS1 protein to interfere with host mRNA processing. Instead, this termination defect is a common effect of diverse cellular stresses and underlies the production of previously reported downstream-of-gene transcripts (DoGs). Our work has implications for understanding not only host-virus interactions but also fundamental aspects of mammalian transcription.  •Influenza virus infection dysregulates host transcription•Viral infection depletes Pol II from gene bodies downstream of the TSS•Virus-induced stress leads to a catastrophic failure of Pol II termination•Defective termination does not require viral NS1: host CPSF30 interaction Bauer et al. investigate the effects of influenza virus infection on host RNA polymerase II (Pol II) transcription genome-wide. They find that infection leads to dysregulation at both the starts and ends of genes. Their work provides insight into both virus-host interactions and fundamental mechanisms of mammalian transcription. Influenza virus remains a challenge for global health and is responsible for both seasonal and pandemic outbreaks . It has The C-terminal domain of the large subunit of host Pol II (CTD) contains multiple repeats of the amino acid heptad: Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, which are post-translationally modified in order to regulate and coordinate the transcription cycle and RNA processing and act as an assembly platform for various accessory enzymes . FluPol Viruses exploit a variety of mechanisms to prevent the host from responding to viral infection and often block transcription and translation of host genes . AlthougAlthough the studies above indicate that influenza virus infection can interfere with the Pol II transcription cycle at multiple stages, the global consequences for Pol II transcription remain unknown. Inhibition of transcriptional elongation and CPA suggest an effect on termination of Pol II, which is a highly coordinated and regulated process . Indeed,Given the intimate association between influenza virus and host transcription, we have explored the dynamics of Pol II during influenza virus infection using mammalian native elongating transcript sequencing (mNET-seq). We show that host transcription is drastically altered as a result of influenza virus infection and find that influenza virus mounts a two-pronged attack on host transcription. First, infection leads to decreased Pol II occupancy downstream of transcription start sites (TSSs), and second, interference with 3′ end processing leads to marked defects in termination of Pol II transcription at the end of genes.To examine the effect of influenza virus infection on host Pol II transcription in human lung epithelial cells, we infected A549 cells at a high MOI (MOI = 5) with an H1N1 influenza virus (A/WSN/33) and carried out mammalian native elongating transcript sequencing (mNET-seq) to provide a single-nucleotide resolution map of Pol II activity genome-wide . ExaminaWe have examined the apparent depletion of Pol II downstream of TSSs in more detail to determine whether the effect is limited to particular genes and to understand the mechanism underlying the defect. We computed the Pol II occupancy  observedWe next turned our attention to the marked defect in host transcription termination during influenza virus infection. Pol II occupancy normally increases immediately downstream of the poly(A) sites of protein-coding genes as a result of Pol II pausing before termination . HoweverWe considered whether the well-studied interaction between the influenza virus NS1 protein and host CPSF30  was respDespite the considerable attention given to the canonical interaction between influenza virus NS1 and host CPSF30, the NS1 proteins of many influenza strains have mutations that prevent their binding to CPSF30 . In orde+ RNA  that arise from continued transcription tens of kilobases downstream of normal poly(A) sites . These D+ RNA . We exam+ RNA A. Remark+ RNA B, and th+ RNA . This te+ RNA C. On the+ RNA D and fou+ RNA . Similar+ RNA E. We con+ RNA  and oxid+ RNA .Figure 6We have analyzed the behavior of host Pol II during influenza virus infection and show that influenza virus executes a two-pronged attack on host transcription, with (1) FluPol association with Pol II leading to depletion of Pol II in gene bodies and (2) viral stress and viral protein-mediated interference of CPA leading to a marked termination defect at normal poly(A) sites. Our results have implications for understanding both viral effects and fundamental aspects of mammalian transcription that have consequences for virus-host interactions.Our observation that influenza virus infection leads to decreased Pol II gene occupancy downstream of the TSS suggests that FluPol association with Pol II can act as a simple mechanism of host shutoff by dysregulating host transcription. In contrast to the termination defect at the end of genes , this depletion of Pol II downstream of the TSS is specific to influenza virus infection. A number of mechanisms could underlie the decreased Pol II occupancy we observe. One model would involve FluPol inducing extremely premature termination of Pol II, perhaps mediated by cleavage of nascent host transcripts by FluPol that renders the exposed 5′ end of the nascent Pol II transcript a suitable substrate for degradation by Xrn2. This would lead to premature termination as a result of Pol II being “torpedoed”  off geneA second model would involve FluPol blocking access of Pol II regulatory factors to Pol II and its CTD . Such a In both cases, dysregulation of Pol II transcription downstream of the TSS will have functional consequences for host shutoff. In the context of a cell in which antiviral gene expression has been triggered, the recruitment of Pol II to these genes will also consequently recruit FluPol, allowing the virus to counteract the activation of cellular defenses. Any subsequent extremely premature Pol II termination that occurs as a result of cap snatching could also benefit viral transcription in cases where Pol II is more rapidly cleared from promoters and gene bodies, because free Pol II would now be free to re-initiate another round of transcription to feed further capped nascent RNA to FluPol. Such a relationship between apparent Pol II depletion in gene bodies and host shutoff is consistent with observations that most infected cells fail to induce a detectable interferon response even in the absence of NS1 . It seemAlthough it appears advantageous for a virus such as influenza to prevent expression of antiviral genes by interfering with CPA, it is also possible that failure to disengage Pol II at the end of genes during influenza virus infection provides a benefit to the host cell. Thus, continued transcription by Pol II might decrease the pool of Pol II available to re-initiate transcription and thereby starve influenza virus of the nascent, capped transcripts it needs to prime its own viral mRNA production. Interestingly, analysis of circulating H1N1 viruses suggests that CPSF30 inhibition is advantageous during adaptation to human  hosts . AdditioStructural analysis of how host shutoff operates is also lacking. Although we know that FluPol binds the Pol II CTD , how FluWe observe multiple effects of influenza virus infection on the behavior of Pol II. First, Pol II is depleted downstream of TSSs as a consequence of influenza virus infection. Intriguingly, although Pol II termination downstream of poly(A) sites usually results in an accumulation of paused Pol II  , the lacSecond, we observe widespread failure of Pol II to terminate downstream of poly(A) sites during both influenza virus infection and cellular stress from osmotic shock. These results suggest that previously observed DoGs arise from CAT. Although the DoGs that have been observed and characterized are polyadenylated and appear downstream of only a subset of genes , the PolThe observed Pol II termination defect during influenza virus infection could restrict the amount capped nascent RNA available to FluPol and therefore be a protective cellular response. However, viral infection might simply be causing general cellular stress conditions that induce a Pol II termination defect or affect pathways related to these conditions. Influenza virus is known to induce oscillations in calcium ion concentrations , which mIt is also possible that the influenza virus-induced termination defect relates to a more general stress response, for example to maintain chromatin structural integrity  during oervin.fodor@path.ox.ac.uk).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Prof. Ervin Fodor  cells were maintained in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal calf serum (FCS). Madin-Darby Bovine Kidney (MDBK) and Madin-Darby Canine Kidney (MDCK) epithelial cells were maintained in Minimum Essential Medium  supplemented with 2 mM L-glutamine and 10% FCS. Human embryonic kidney 293TRex/NS1 and 293TRex/NS1mut cell lines  were maiFor infection experiments, A549 cells were grown to 70%–80% confluency in 15 cm dishes, and infected at an MOI of 5 in DMEM. For all experiments, uninfected control cells were included which were mock-infected using DMEM. Cells were harvested at 12 hours post-infection for all viruses except WSN, which was harvested at 6 hours post-infection. Cells were harvested by washing with 20 mL of ice-cold PBS, collected by scraping, and transferred to 15 mL tubes. Cells were pelleted by centrifuging at 1000 x g at 4°C for 5 minutes, aspirating the supernatant, and flash-frozen in liquid nitrogen. Samples were stored at −80°C until use. Infections of 293TRex/NS1 cells with WSN were also carried out at an MOI of 5, with cells harvested at 4.5 hours post-infection by scraping first, then pelleting and resuspending twice in ice-cold PBS before flash-freezing in liquid nitrogen. For NS1 expression experiments, culture media was supplemented with 1 μg/ml tetracycline (Sigma) and cells were harvested after 24 hours. Osmotic shock experiments were carried out as described  by supplin situ, extracted using TRIzol (Invitrogen), and size-selected on a denaturing polyacrylamide gel to obtain fragments 35-100nt long. For mNET-seq, 293TRex/NS1 cells were prepared in the same manner, except micrococcal nuclease digestion was performed for 140 s, and RNA was isolated following 3′ phosphorylation using the Quick-RNA microprep kit (Zymo Research) and 17-200nt RNA was size-selected according to the manufacturer’s protocol.Sample preparation for mNET-seq was performed as previously described . BrieflyLibraries suitable for Illumina sequencing were prepared from RNA extracted following mNET-seq sample preparation using the TruSeq Small RNA Library Preparation kit (Illumina) for A549 cells, or the NEBNext Small RNA Library Preparation kit (NEB) for 293TRex/NS1 cells. Sequencing libraries were amplified by 15 cycles of PCR and size-selected on a 6% Novex TBE PAGE gel according to the manufacturers’ instructions to remove primer-dimers. Paired-end sequencing (2x75bp) was performed on an Illumina HiSeq 2500 in Rapid Mode, or an Illumina HiSeq 4000 instrument at the Oxford Genomics Centre, Wellcome Trust Centre for Human Genetics .cutadapt 1.8.3  site and the Pol II occupancy in the 2kb upstream of the poly(A) site. Genes with less than 10 reads in either case were excluded from analysis. A long-range Read-Thru Index was calculated by taking the log2 ratio between Pol II occupancy in the 2kb upstream of the poly(A) site and the Pol II occupancy in the interval poly(A) site+8kb to poly(A) site+10kb. The level of Pol II occupancy and the statistical indices above were compared between samples, and P values computed, using the Wilcoxon Rank Sum (Mann-Whitney) Test via the wilcox.test function in R. Published lists of DoG transcript genes during osmotic shock  . Metagenescribed . A Read-ic shock  were useThe accession number for the sequencing data reported in this paper is NCBI SRA: SRP132032.


Add to this the fact that we have no idea how many different species of viruses there actually are. He then segued into the topic of virus quantification and that many of the current methods for quantifying viruses are outdated, expensive and highly subjective. For example, the plaque titer assay is essentially the same as it was when first used in 1952. Infectivity assays in general tend to be fairly slow, taking anywhere from 3–14 d to obtain results depending on the virus of interest. More recent technologies such as transmission electron microscopy are very costly, difficult to use and do not differentiate between empty and intact viral particles. So, how does this work in today's environment especially when someone is using emerging approaches such as Virus-Like Particles and what can we do to improve quantification? This is a problem that extends throughout many industries, especially during the production of viruses.Mark Rehse form ViroCyt opened his talk with the question of how many viruses are in the ocean as a way of introducing the topic of virus quantification. As it turns out, there is an estimated 100 million viruses in a milliliter of your average sea water, which—if you do the math—means that there are 1.3 × 10Vaccine manufacturing was one the first applications that ViroCyt focused on improving processes by using true quantitative technologies. A significant amount of work has also been done with baculovirus expression systems, antiviral drug development, and more recently, viral therapeutics or so-called oncolytic virocides. It's a huge, growing industry in the United States, generally in teaching hospitals, where they have viral vector core labs and they’re taking retroviruses, vaccinia viruses—creating a variety of oncolytic viruses—by engineering them to deliver a message to a target in the solid tumor. Incredibly interesting stuff and very effective, but they all lack one thing, which is a new technology, a modern technology to accurately and quickly quantify viruses. As a result, ViroCyt created the Virus Counter®. The Virus Counter 3100 is the newest addition to ViroCyt's portfolio of products, and includes a standalone component as well as an optional 96-well plate autosampler , an interdepartmental and interagency group established to support and facilitate the creation of vaccines and therapies for Ebola and Marburg viruses. It is co-led by the Department of Defense and the Department of Health and Human Services, and includes participation by the governments of Canada and Great Britain.Taking closer look at the instrument, the software interface for the autosampler coupled system indicates which well is currently being analyzed, which wells have been completed as well as those that have been programmed for quantification during the sequence. In the automated system, the liquid handling for both supply and waste has also been scaled up permitting up to 15 hours of continuous operation.He went on to present several case studies as specific examples of how the Virus Counter is being used today, the first focusing on baculovirus. The applications of baculovirus—since it was first developed as an expression system in the early 1980s—have grown tremendously and it is now used for all sorts of expression vector programs and in final product manufacturing. The Virus Counter fits quite well into many aspects of the baculovirus system, such as the expansion phase, purification, and even in the determination of which construct produces the highest titer. These are at least 3 steps that can benefit from a way to measure total virus construct. Recently acquired data from a clinical site in the United States looked at 2 different types of baculovirus constructs: One designed to express GFP and the other AAV. There were 2 different concentrations of each of the constructs which were followed over a 60 hour growth period, and there is a dramatic difference between the AAV-producing component and the GMP-expressing component in terms of growth output. Mark then asked, “How many samples would it take to generate this data using traditional plaque titer assays? With the answer being quite a while. It raises the question of do we really want to spend the time, energy and money to do this, or should we just choose one option and go with it?” Using the Virus Counter provides the advantage of being able to make a decision on a number of constructs simultaneously, since there could have been 3 or 4 or 5 more constructs evaluated and take the same amount of time to generate the data.Mark then mentioned that the staining protocol takes 30 minutes at room temperature in the dark. The dyes are intercalating equilibrium dyes and not antibody-based, so there is no wash step involved, and one minute to acquire the data.In the next use example, a set of 13 different baculovirus constructs were evaluated using TCID50 and the Virus Counter (is infectious, one can see that-during the purification process-the Fluorescent Focus Units decrease while the total particle count increases or plateaus. This strongly suggests that the VLPs are fractioning into the centrifugation product while the baculovirus is spun down and separated out. A second series of experiments diagrammed an ultracentrifugation experiment comparing the supernatant and pellet for VLP content (VLPs were the focus of the next group of results. Since VLPs are non-infective, many traditional approaches to quantification are not useful. As mentioned previously, the Virus Counter can replace OD495 measurements and is much faster and more reproducible. In work carried out by a collaborator, the enrichment of VLPs generated by a baculovirus expression system was tracked using Virus Counter and the Fluorescent Focus Assay (In summary, the Virus Counter requires a minimum sample volume of 100 microliters, has a detection limit of 1×10E5 to 1×10E9 vp/ml, and the upper limit can be addressed using serial dilution of concentrated samples. The total analysis time is 6 minutes, but this can be decreased depending on the nature of the samples. It takes 1 minute to quantify the actual sample, followed by an intersample wash using a low pH solution and verification that there isn't any residual carryover from the previous sample.


We report draft genome sequences of three novel bunyaviruses, two novel rhabdoviruses, and one novel nyamivirus identified metagenomically from 10 moths in Washington state. Noctua pronuba, Tyria jacobaeaea, the timber pest Orgyia pseudotsugata (two sampled), and the apple pests Pasiphila rectangulata, Carcina quercana, Plutella xylostella, Choristoneura rosaceana, and Cydia pomonella (two sampled)  was assembled corresponding to the novel rhabdovirus Orgi virus SP1. The L polymerase protein aligned 45% by amino acid to the Wuhan Fly Virus 2  that aligned 99.6% by nucleotide to the original Orgi virus SP1. An additional 11,238 nucleotide contig with five ORFs was assembled from this sample corresponding to the novel rhabdovirus Gata virus M4. Its RdRp aligned closest to the Orgi virus at 47% by amino acid.From one  Virus 2 . SequencCydia pomonella sequenced, three segments corresponding to the RdRp, glycoprotein, and nucleocapsid/ORFIV segments of Mothra bunyavirus JG1 were assembled. Interestingly, sequencing of an additional Cydia pomonella revealed 170× coverage of the Mothra bunyavirus RdRp and nucleocapsid segments but no reads to the glycoprotein segment. The RdRp of the two Mothra bunyaviruses shared 97.3% amino acid identity and aligned 28% by amino acid to the RdRp Uukuniemi virus (AIU95041) isolated from Ixodes ricinus from the Czech Republic , KX852389 to KX852391 (Pidgey bunyavirus M6), KX272883 to KX272885 and KY293690 to KY293691 (Mothra bunyavirus JG1), KX272880 to KX272882 (Seattle Prectang UW1), KX852388 (Gata virus M4), and KX852386 to KX852387 .These genomes have been deposited at DDBJ/ENA/GenBank under the accession numbers


Rat somatosensory cortex contains a large sexually monomorphic genital representation. Genital cortex undergoes an unusual 2-fold expansion during puberty. Here, we investigate genital cortex development and female rat sexual maturation. Ovariectomies and estradiol injections suggested sex hormones cause the pubertal genital cortex expansion but not its maintenance at adult size. Genital cortex expanded by thalamic afferents invading surrounding dysgranular cortex. Genital touch was a dominant factor driving female sexual maturation. Raising female rats in contact with adult males promoted genital cortex expansion, whereas contact to adult females or nontactile  male cues did not. Genital touch imposed by human experimenters powerfully advanced female genital cortex development and sexual maturation. Long-term blocking of genital cortex by tetrodotoxin in pubescent females housed with males prevented genital cortex expansion and decelerated vaginal opening. Sex hormones, sexual experience, and neural activity shape genital cortex, which contributes to the puberty promoting effects of sexual touch. We recently identified the somatosensory representation of rat genitals; remarkably, this cortical region—genital cortex—is sexually monomorphic, despite the marked sexual dimorphism of external genitals in rats. Most intriguing was the observation that genital cortex doubles in size during puberty. In order to shed light on this unusual expansion, we studied genital cortex development and sexual maturation in the female rat. We first showed that sex hormones are likely to cause the pubertal expansion of genital cortex. Next, we examined whether sexual experience affects the development of female genital cortex. Raising females together with adult males advanced genital cortex expansion, but cohousing with adult females or exposure to nontactile male cues was not sufficient to drive genital cortex growth. Surprisingly, artificial genital touch led to an early onset of female puberty and growth of genital cortex. In line with this finding, we find that if genital cortex activity is blocked, the advancing effects of adult males on puberty and genital cortex growth are inhibited. Together, our results point to an important role of genital cortex in the puberty-promoting effects of sexual touch. Early analysis of the development of visual cortex by Hubel and Wiesel focused on binocular interactions and showed that both anatomy and physiology of ocular dominance is plastic and shaped in a visually-driven, activity-dependent process . In a siWe were also interested in the relationship between somatosensory genital cortex development and female sexual maturation. In rodents, puberty has been intensely studied and much is known about changes in subcortical structures, such as the hypothalamus, that are associated with puberty ; there iIn primates, the evidence for pheromonal effects on puberty is rather mixed . Sexual Here we ask the following questions: (1) Are sex hormones required for the pubertal expansion of female genital cortex? (2) What are the structural events underlying genital map changes? (3) Does sexual experience affect the development of female genital cortex? (4) Does activity in female genital cortex affect female puberty and does it mediate the female puberty advancing effects of male sexual touch?P < 0.01, Student t test). In contrast, the size of the entire S1 did not differ significantly between young and old females .As a first step in our analysis we performed additional experiments to confirm our recently reported data on the growth of genital cortex during puberty . To thisP < 0.01, Student t test) smaller than the genital cortex size in animals, which were ovariectomized after puberty  and after puberty (at P42). Twenty days after the ovariectomy animals were killed, brains were removed and tangential sections through the S1 were prepared and stained for cytochrome oxidase activity . We obtaWe conclude that sex hormones are required for the pubertal expansion of female genital cortex but not for the maintenance of an adult size female genital cortex.n = 7 animals; 0.05μg estradiol, dissolved in 0.4 ml of sesame oil, per 100 g body weight) or with sesame oil alone . During the fifth day of treatment animals were killed and cortical hemispheres were flattened, tangentially sectioned and processed for cytochrome oxidase activity  and the mean percentage of genital cortex of S1  was significantly greater in estradiol injected animals compared with control animals (oil treated). The size of S1 was not different .The lower panel in P < 0.001, Mann–Whitney U test). We also removed and weighed the uteri. On average, the oil-treated animals had lighter uteri . Taken together these data suggest that systemic estradiol accelerates genital cortex growth and advances the onset of puberty.We also assessed parameters indicative of sexual maturation and the onset of puberty. Thus, we evaluated vaginal opening  and theWhich cellular changes underlie the unusual genital cortex expansion during puberty? To answer this question, we first stained alternating tangential sections of flattened cortices for cytochrome oxidase activity  and with2 ± 0.06 and in old animals 0.56mm2 ± 0.1). Interestingly, the ratio of the genital cortex to the interlimb cortex significantly increases during puberty . The simultaneous increase of genital cortex and decrease of interlimb cortex suggest that the pubertal genital cortex growth reflects an invasion of dysgranular interlimb cortex by putative genital thalamic afferents.Next, we asked, how the genital cortex is able to increase its thalamically innervated area so drastically. Does the genital cortex expand like a balloon, pushing the neighboring cortex aside? Alternatively, do genital afferents invade neighboring territories? To address this issue, we analyzed S1 maps from young and old animals in more detail . SpecifiAlthough sex hormones have been identified as important players during the onset of puberty, there is also evidence that somatosensory stimuli (olfactory or tactile) can influence sexual development. Thus, we assessed if sexual experience affects the development of female genital cortex. To this end, we chose the following experimental paradigm, which is inspired by the seminal work of Bronson and Maruniak . PrepubeP = 0.01). The overall size of S1 . Note that the relative size of genital cortex of the young females, which were cohoused with sexually experienced males  and the relative size . The difference between hemispheres from control  and TTX treated animals  was marked . These results immediately suggest 2 conclusions. First, the fact that there was a difference between control and TTX treated animals indicates that our TTX treatment was effective. Second, the data indicate that the pubertal expansion of genital cortex requires cortical activity.In order to better understand the role of female genital cortex during puberty, we blocked genital cortex activity over a certain time during prepuberty P23–P30). To do so, P21 animals received Elvax implants over the area of genital cortex. Elvax sheets were developed for the slow, gradual release of drugs [–P30. To ive size  was smaln = 11), whose genital cortices were blocked with TTX, had an open vagina. Next, we wondered if an intact genital cortex is required for female sexual maturation. Therefore, we determined the status of puberty in control and TTX treated animals. Accordingly, pictures of vagina and clitoris were taken before implantation and after the behavioral experiment. Five out of 13 animals, which received a ringer soaked Elvax sheet above genital cortex, showed an open vagina at the end of the experiment. The vagina before (left panel) and after the experiment (right panel) for one of those females is shown in In order to obtain insight in the relationship between female sexual maturation and genital cortex development, we plotted genital cortex size against uterine weight in all experiments, in which we collected such data . As showOur data confirm a major pubertal expansion of female genital cortex, which—unlike the maintenance of large genital cortex in adults—requires intact ovaries and sex hormones. The larger area of genital cortex in adults reflects an invasion of adjacent territories by thalamic afferents. Such changes in female genital cortex are advanced by sexual touch and require cortical activity. Most interestingly, blockade of genital cortex delays female sexual maturation, suggesting that genital cortex is the neural structure that mediates the puberty advancing effects of male sexual touch.The expansion of layer 4 of genital cortex in puberty  is an unThe data discussed below indicate that at least some stage of cortical activity seems to be required for genital cortex expansion. The visualization of thalamic afferents by VGluT2 antibodies showed that cytochrome oxidase staining reveals maps completely congruent with thalamic innervation. Thus, the size increase of genital cortex in adults indicates that a larger cortical area is innervated by thalamic afferents. The detailed analysis of body maps in young and adult animals is strongly suggestive that the size increase of genital cortex results from an invasion scenario, in which putative genital afferents innervate neighboring dysgranular cortex.Female genital cortex development is strongly affected by tactile sexual experience. Housing pubescent females with a sexually experienced adult male strongly advances the growth of genital cortex. Even though we killed such females at P30 , they had genital cortices larger than average adult size .pm. Rats had ad libitum access to food and water.All experiments were conducted on Wistar rats purchased from Janvier Labs. All animals were kept on a 12 hour to 12 hour, normal light/dark cycle with lights off at 10:00 Ovariectomies were performed in prepubescent (P21) and adult Wistar rats (P42). For the surgery, animals were anesthetized by injection of an initial dose of 100 mg/kg ketamine and 7.5 mg/kg xylazine. Respiration, blink, and pinch reflex were observed throughout the surgery and, if needed, animals were injected with an extra shot (25%) of ketamine/xylazine mixture  or a 25% dose of ketamine (Sigma-Aldrich) alone. Monitoring of temperature was done using a rectal probe and could be maintained with a heating pad  to 34°C–36°C.To remove ovaries a small incision was made bilaterally on the animal’s bag. The white fat pad, to which the ovaries are attached to, was pulled out through the body wall. Ovaries were grabbed with forceps and removed from the fallopian tube using small scissors. After successful removal, the white fat pad with fallopian tube was put back through the body wall. The incision was sutured using a self-resorbing thread. The wound was disinfected and looked after in the days following surgery.Immature Wistar rats (P26) were divided randomly into groups of 3–4 animals. All animals were weighed daily and the vagina opening was assessed by visual inspection and defined as a complete separation of the membranous sheath covering the vaginal orifice . Photos Animals in the testing group were injected subcutaneously every morning with 0.05 μg 17β-estradiol (Sigma-Aldrich), dissolved in 0.4 ml of sesame oil (Sigma-Aldrich), per 100 g body weight. Rats in the control group received the same amount of sesame oil. On day 5 of the treatment, animals were deeply anaesthetized, perfused, and their brains processed, as described below. In addition, uteri of every animal in experiment was taken out while perfusion and weighed.Wistar rats aged P21 were divided into 3 groups. Each animal of group 1 was housed for 9 days together with a sexually experienced adult female rat (approximately P60). Every rat of group 2 was put for 9 days together with a sexually experienced adult male rat (approximately P60). Group 3 rats were also housed together with a sexually experienced adult male rat (approximately P60), but the cages were separated by a wire-mesh. Whereas the animals of group 1 and 2 were able to interact fully with their housing partner, the rats in group 3 could not touch the male they were housed together with. However, they were able to smell, see, and hear the male through the wire-mesh.Vaginal opening was documented as described above, and pictures of clitoris and vagina were taken before and after the experiment. After 9 days, at the age of P30, animals of all groups were anaesthetized, perfused, and their brains histologically processed as described below. Uteri of all animals were taken out while perfusion and weighed.Prepubescent female Wistar rats (P23) were divided into 2 groups. Control animals were placed 3 times for 10 minutes in a small U-shaped arena over 7 days without further treatment and were then returned to their home cage. Animals in the artificial genital touch group were placed 3 times for 10 minutes in a small arena over 7 days, and their clitoris and vulva were contacted with a lubricated brush by a human  experimenter . After Animals (P21) were divided into 2 groups. The control group received a chronic Ringer-impregnated Elvax sheet above genital cortex and the rats in the testing group were implanted with a TTX-impregnated Elvax sheet above genital cortex.Elvax implants were prepared as described before , 37, 38.For surgical implantation P21 Wistar rats were anesthetized by injection of an initial dose of 100 mg/kg ketamine and 7.5 mg/kg xylazine. Respiration, blink, and pinch reflex were observed throughout the surgery and, if needed, animals were injected with an extra shot (25%) of ketamine/xylazine mixture or a 25% dose of ketamine alone. Monitoring of temperature was done using a rectal probe and could be maintained with a heating pad to 34°C–36°C. Lidocaine was locally injected in the scalp, which was then removed. A craniotomy was performed above genital cortex, and the dura was removed using a bend syringe. The TTX or Ringer impregnated sheets were placed on the brain surface and covered with silicone . The exposed skull was finally covered with dental cement. Animals were allowed to recover from surgery for 2 days. Photos of clitoris and vagina were taken before the behavioral experiment. At the age of P23, implanted animals were put together with a sexually experienced male. After 7 days (at the age of P30), TTX and control animals were anaesthetized and their vaginal opening was documented as described above. Along with the perfusion, the uteri of the animals were cut out and weighed. Brains were histologically processed as described below.At the end of the above-described experiments, animals were anaesthetized using a 20% urethane solution and perfused with phosphate buffer followed by a 2% paraformaldehyde solution (PFA). Brains were removed, hemispheres were separated, and cortices were flattened between 2 glass slides separated by clay spacers . Glass sFor the alternating staining procedure with cytochrome c and VGluT2, immunohistochemical labeling was performed using standard procedures. Briefly, brain sections, which should be labeled for VGluT2, were preincubated for an hour at room temperature in a blocking solution . Afterwards, primary antibodies were diluted in a solution containing 0.5% Triton X-100 and 1% bovine serum albumin. The primary antibody against VGluT2 was incubated with the free-floating sections for at least 24 hours under mild shaking at 4°C. Incubations with the primary antibody was followed by detection with a secondary antibody coupled to the fluorophore Alexa 488. The secondary antibody was diluted (1:500) in 0.5% Triton X-100 and the reaction was allowed to proceed for 2 hours in the dark at room temperature. After the staining procedure, sections were mounted on gelatin coated glass slides with Mowiol mounting medium.The area of various somatosensory regions was measured by outlining the anatomical region of interest and calculating its area using Neurolucida area calculating tool. The area of the following cortical representations was measured: hindpaw, forepaw, trunk, interlimb cortex, and clitoris. The fraction of genital cortex of the whole S1 area was calculated by dividing the clitoris area by the value of the S1 area. The same was done for the fraction of genital cortex of the interlimb cortex. All statistic tests were conducted in Matlab.In order to quantify the state of vaginal opening after the above described experiments, we assigned different states with vaginal opening scores. A score of 0 points to a closed vagina, whereas a score of 0.5 describes a vagina, which was about to open. Finally, a score of 1 represents an open vagina.S1 FigA, Upper panel: Tangential section through S1 of a P25 old animal stained for cytochrome oxidase activity. Lower panel: The corresponding map drawn from the section shown above, genital cortex shown in black.B, Same as A, but the section and map stems from an animal aged P42. The genital cortex is larger than in the P25 animal. C, Same as A for an animal aged P42, which was ovariectomized before puberty at P20. Note that the size of the genital cortex is comparable to the genital cortex of the youngfemale shown in A. D, Same as A for an animal aged P60, which was ovariectomized after puberty at P42. The clitoris representation is greater than in the young ovariectomized animal (C) but comparable in size to the P42 aged animal (B).Figure S1 related to (TIF)Click here for additional data file.S2 FigA, Fraction of genital cortex of the entire S1 in hemispheres of P25, P42 females and females which were ovariectomized at either P20 or P42. B, Same as A but fraction of genital cortex of the PMBSF is shown. Note that the same effects are seen as in A, namely a substantial growth of the genital cortex between P25 and P42 animals. Female rats ovariectomized during prepuberty had smaller genital cortices than animals ovariectomized after puberty. Graphically distribution looks similar in A and B and no big difference in the variability can be make out. See also C, Absolute area of PMBSF in hemispheres of P25, P42, in prepuberty (P20) ovariectomized and postpuberty (P42) ovariectomized female rats. See also Figure S2 related to (TIF)Click here for additional data file.S3 FigA, Upper panel: Tangential section through S1 of a hemisphere obtained from an animal which received subcutaneous sesame oil injections over 5 days. The section was stained for cytochrome c and shows the reconstructed clitoris area best. Lower panel: Corresponding reconstructed map from the section plotted above. B, Same as A, but the section and drawn map are obtained from an animal which received daily estradiol injections. Note that the clitoris area is greater compared to the example section and map shown in A.Figure S3 related to (TIF)Click here for additional data file.S4 FigA, Upper, tangential section through S1 of an animal aged P14. The section was stained for cytochrome oxidase activity. Lower, map of the somatosensory areas drawn on the section shown above. B, Upper, adjacent tangential section through S1 obtained from the same hemisphere, which is shown in A. The section was stained with antibodies (green fluorescence) against VGluT2 (vesicular glutamate transporter 2), which is expressed in thalamocortical afferents. Lower, map of the somatosensory areas drawn on the section shown above. Note that the reconstructed areas correspond in size when comparing A and B.Figure S4 related to (TIF)Click here for additional data file.S5 FigA, Genital cortex size is plotted against uterine weight for all experiments. No tight relationship can be observed. Note that a large uterine weight (≥ 0.15 g) is rarely associated with a small (≤ 1%) relative genital cortex size.B, Same as A, but for specific experimental conditions See also Figure S5 related to Figs (TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S1 DataIndividual data for Figs (XLSX)Click here for additional data file.


Zika virus is an enveloped, positive-strand RNA flavivirus. It was first isolated in 1947 from the Zika forest of Uganda from a Rhesus macaque. After that for many years, it was not known whether the virus can cause human disease. The epidemiology of Zika virus changed since 2007 when an outbreak occurred on Yap Island of the Federated States of Micronesia. Zika virus infection generally leads to self-limiting mild, febrile illness. However, many of the recent outbreaks were linked to upsurge in cases of GuillanBarré syndrome (GBS) and a rise in infants born with microcephaly. Placenta and amniotic fluid of women with microcephalic fetuses and blood of neonates with microcephaly were found positive for Zika virus RNA suggesting the transplacental transmission of Zika virus. The virus has also been identified in the brains and retinas of microcephalic fetuses (Despite accumulating clinical evidence, direct experimental evidence showing that the Zika virus causes birth defects remains absent. Zika virus infects fetuses, is causing intrauterine growth restriction, including signs of microcephaly, in mice . Zika viZikavirus and man share a peptide in common, hence, immune response to Zika virus might cause damage to host tissue and responsible for all the neurological complications . Along w


Asp-Glu-Ala-Asp (DEAD)-box polypeptide 5 (DDX5), also called p68, is a prototypical member of the large ATP-dependent RNA helicases family and is known to participate in all aspects of RNA metabolism ranging from transcription to translation, RNA decay, and miRNA processing. The roles of DDX5 in cell cycle regulation, tumorigenesis, apoptosis, cancer development, adipogenesis, Wnt-β-catenin signaling, and viral infection have been established. Several RNA viruses have been reported to hijack DDX5 to facilitate various steps of their replication cycles. Furthermore, DDX5 can be bounded by the viral proteins of some viruses with unknown functions. Interestingly, an antiviral function of DDX5 has been reported during hepatitis B virus and myxoma virus infection. Thus, the precise roles of this apparently multifaceted protein remain largely obscure. Here, we provide a rapid and critical overview of the structure and functions of DDX5 with a particular emphasis on its role during virus infection. Escherichia coli to humans . Si. Si18]. V genome . HoweverV genome . FurtherV genome . This fiV genome . The knonslation . So far,Flavivirus, is one of the common causes for epidemic encephalitis in humans and animals. The resulting disease, JE, affects large parts of Asia and the Pacific, where over 3 billion people at risk of exposure to the virus [Japanese encephalitis virus (JEV), a mosquito-borne neurotropic he virus ,82. JEV he virus . Of theshe virus . The sevhe virus .Recently, it has been shown that DDX5 can interact with JEV core protein (C), NS3 and NS5, and can host DDX5 act as a positive regulator for JEV replication . Both knIn addition to DDX5, helicase DDX3 was also found to be involved in JEV replication . Viral NFlaviviridae family were also found to hijack DDX5 during virus infection. The genus Pestivirus, a group of small positive-stranded RNA viruses, belongs to the family Flavivirus, which cause economically important diseases of farm animals and includes bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), and border disease virus (BDV) [pro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS5B-COOH [pro, named for the viral N-terminal protease, is a 168-amino-acid autoprotease, with cysteine protease activity in a Glu22-His49-Cys69 triad that acts to cleave itself from the nascent polypeptide [pro binds to DDX5, and also to DDX3X and DHX9 [pro in ribosomal and ribonucleoprotein particles, as well as its interactions with several RNA helicases, suggest that Npro is involved with virus RNA translation; DDX5 possibly contributes to this process. Nevertheless, the function of DDX5 in modulation of viral replication remains to be determined.Besides HCV and JEV, other members of the us (BDV) . The envS5B-COOH ,91. Nproypeptide . Early wand DHX9 . The disArterivirus of the family Arteriviridae [Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of one of the most economically important global swine diseases, is classified in the genus iviridae . PRRSV civiridae . ORF1a aiviridae .Among the nsps encoded by the ORF1b region of PRRSV, nsp9 is considered to be involved in viral replication and genomic transcription . This prConfocal immunofluorescence assay has revealed that DDX5 co-localizes with nsp9 in the cytoplasm with a perinuclear pattern in HEK293 cells, MARC-145 cells, and the PAM cell line, suggesting that endogenous DDX5 might diffuse from nucleus to cytoplasm and help to unwind the viral RNA through its helicase activity. This hypothesis was confirmed by co-immunoprecipitation (Co-IP) assay, which showed that the DEAD and helicase domains (aa1-436) of DDX5 bind with the C-terminal portion of nsp9 . InteresInfluenza A virus is a major public health problem, causing seasonal epidemics in humans and occasionally lethal global pandemics, as happened in 1918–1919 (H1N1), 1957 (H2N2), 1968 (H3N2), and 2009 (swine-origin H1N1) . CurrentIt has been demonstrated that the trimeric viral polymerase  and nucleoprotein (NP) contribute to the pathogenicity of H5N1 viruses in humans and other species of mammals . FunctioEpstein-Barr virus (EBV), a type of oncogenic human herpesvirus, is ubiquitous, causing over 90% infection in the exposed human population . InfectiLeporipoxvirus genus, Myxoma virus exhibits a specific rabbit-restricted host tropism but reveals a much broader cellular host range in cultured cells [Being a member of the ed cells . MYXV haed cells . Recentled cells . In addied cells . Howevered cells .Additionally, DDX5 has a role in transcription of HBV minichromosome by a mechanism not yet determined . HBV is In recent years, as the emerging roles of some RNA helicases in host innate response have been established, studies have shown that viral nucleic acids are sensed by DDX1, DHX9, DDX21, DHX33, DHX36, DDX41, retinoic acid-inducible gene 1 (RIG-I/DDX58), and melanoma differentiation- associated gene 5 (MDA-5), leading to type I interferon production and an antiviral response ,120,121.As summarized in this review, the multifaceted protein DDX5 has been shown to be involved in RNA metabolism and viral infection, especially for RNA viruses. It is intriguing that DDX5 is the target of manipulation by different viruses. Each RNA virus seems to hijack host DDX5 in order to facilitate its own replication. With conserved ATP binding motif and helicase motif, DDX5 utilizes the free energy to change of binding and hydrolyzing a nucleotide triphosphate to dissociate duplexes or displace bound proteins, which, on one hand, DDX5 may translocate along with viral DNA/RNA; on the other hand, DDX5 may unwind double-stranded regions to promote the expression of viral proteins . HoweverEven though emerging studies on DDX5 have supplemented our knowledge about the multiple functions of DDX5 in recent years, the overall picture still remains fragmentary. Research is still needed to resolve seemingly contradictory data on the role(s) of DDX5 in response to different virus. As conserved motifs in the primary sequence, DExD/H helicases are highly conserved from viruses and bacteria to humans, and these proteins have overlapping functions. In addition, these proteins generally act as components of large multi-protein complexes, with each other or other factors, to regulate transcription. Therefore, studies should also attempt to establish whether the effects of DDX5 on viral replication after knockdown are unique or whether other helicases supplement abolished function via small interfering RNA.


Gastric cancer is one of the leading causes of cancer-related deaths in both sexes worldwide, especially in Eastern Asia. This study aimed to estimate the economic burden of advanced gastric cancer (AGC) in Taiwan.n = 122 with AGC) and national statistics. Annual per-patient costs, where patients’ follow-up periods were adjusted for, were estimated with 82 patients who had complete resource use data. The costs were composed of direct medical costs, direct non-medical costs , morbidity costs, and mortality costs. Relevant unit costs were retrieved mainly from literature and national statistics, and applied to the resource use data. A broad definition of morbidity and mortality costs was employed to value the productivity loss in patients with unpaid employment, economically inactive and unemployed as well as the life years after the age of retirement. Their narrow definitions were also used in sensitivity analyses, using age- and/or sex-specific employment rates. Forgone future earnings/productivity loss were discounted at 3%. Annual per-patient costs were projected to estimate the total costs of AGC at the national level with an estimated number of patients with AGC (N = 2611) in Taiwan in 2013.The costs of AGC in 2013 were estimated using resource use data from a chart review study (The mean age of the 82 patients was 59.3 (SD: 11.9) years, and 67.1% were male. Per-patient costs were US$26,431 for direct medical costs, US$4669 for direct non-medical costs, US$5758 for morbidity costs, and US$145,990 for mortality costs (per death). These per-patient costs were projected to incur total AGC costs of US$423 million at the national-level. Mortality costs accounted for 77.3% of the total costs, followed by direct medical costs (16.3%), morbidity costs (3.6%), and direct non-medical costs (2.9%).AGC was found to exert a significant economic burden in Taiwan, incurring US$423 million in 2013. This represents about 0.08% of the Taiwanese economy. Mortality costs appeared to be the single greatest contributor to the burden, followed by direct medical costs. Early detection and providing effective treatments will help to reduce its burden on patients, caregivers and society as a whole.A poster of this study was presented at the 2016 American Society of Clinical Oncology (ASCO) Gastrointestinal Cancers Symposium in San Francisco, CA, USA. Despite a substantial decline in both incidence and mortality over the past decades, gastric cancer (GC) still remains an important public health burden worldwide . It is tGC is characterised by the high variability of non-specific symptoms such as dyspepsia, weight loss and anaemia, which makes early diagnosis difficult. Once GC is detected, it is typically at an advanced stage, making treatments even more burdensome and costly. Notably, the 5-year survival rate at this stage is reported to be less than 25% , resultiA limited number of studies have demonstrated a substantial economic burden of GC in both Western and non-Western countries. For instance, Haga et al. estimated the cost of GC in Japan, using national statistics . They reThis study therefore aimed to quantify the economic burden of GC at both patient-level and national-level in Taiwan; this will help better prioritise and allocate resources for cancer management. Given that GC is often detected at an advanced stage and treatment varies with stage, this study focused on the burden associated with advanced gastric cancer (AGC), using data from a chart review study and national statistics.The present study estimated the economic burden of AGC in Taiwan. There is, however, no single definition of AGC uniformly used in the literature . This stn = 37). Each participating physician was asked to provide de-identified patient-level data from medical charts for up to 10 randomly selected patients diagnosed with metastatic and/or locally recurrent, unresectable gastric cancer , including cancer of the stomach and gastroesophageal junction (GEJ) with adenocarcinoma histology. Data were collected from 1 April 2013 to 8 July 2013 using a secure online chart abstraction instrument.The main data source for this COI study was a retrospective chart review study conducted in Taiwan, which provided data on treatment patterns and resource utilisation among 122 patients with AGC . PhysiciPatients were eligible for inclusion in this chart review study if they (1) had a diagnosis of AGC, including GEJ with adenocarcinoma histology, on or after 1 January 2009; (2) had received first-line treatment with platinum/fluoropyrimidine after AGC diagnosis, were eligible for second-line therapy, and initiated either second-line therapy or best supportive care (BSC) after the first-line treatment; (3) were aged at least 18 years old at the diagnosis of AGC; (4) were not participating in other clinical trials  after AGC diagnosis; and (5) did not have other primary malignant tumours.The details of this chart review study have been published elsewhere .The economic burden of AGC in 2013, expressed in US$ (the market exchange rate of 1 US$ = 30 NT$ ), was esn = 118). Although this is an arbitrary cut-off point, this can help more accurately estimate “annual” per-patient costs, minimising the loss of eligible patient observations. Different follow-up periods were also adjusted for to estimate the annual costs. The final cost analysis was, however, based on 82 patients who had complete resource utilisation data. The per-patient costs were multiplied by the prevalence of AGC in 2013. Given the lack of prevalence data however, the number of patients with AGC in 2013 (N = 2611) were derived from the number of patients with GC in 2012 (N = 4609) and percentages of stage III (27.19%) and stage IV (29.47%) of newly diagnosed GC cases in 2012 in Taiwan ). Nevertheless, the level of resource utilisation was largely similar when comparing the items with all available observations and the items with complete resource utilisation data (data not shown).The total cost of AGC was estimated based on per-patient costs and prevalence of AGC in 2013. The full economic burden of AGC appeared to be substantial with US$423 million at the national-level Table . Per-patTable This study estimated the economic burden of AGC in Taiwan at both patient-level and national-level in 2013. AGC was found to exert a significant economic burden in Taiwan, incurring a total of US$423 million at the national-level in 2013. This estimate represents 0.08% of the Taiwanese economy .There are very few COI studies that have estimated the economic burden of GC in other countries , 9, and While our estimate confirms a substantial burden of AGC in Taiwan, the burden, as a share of GDP, appears to be smaller than those reported in other neighbouring countries like Japan and South Korea where the prevalence of GC is particularly high. For instance, Haga et al. estimated the cost of GC in 1996, 2002, and 2008 in Japan, using national statistics . As in oThere is also a Korean study, which estimated the economic burden of cancer in 2009 , which iAs aforementioned, mortality costs were found to be the single greatest contributor to the AGC burden in Taiwan. The costs accounted for more than two-third of the total costs (77%), followed by direct medical costs (16%), morbidity costs (4%), and direct non-medical costs (3%). These findings indicate that improving survival of GC patients can substantially reduce the economic burden of AGC in Taiwan. Notably, Taiwan has a mortality-to-incidence ratio of about 0.59–0.63 –34. ThisWhile premature death associated with AGC were estimated to be the costliest component of AGC burden in Taiwan, these mortality costs (as well as morbidity costs) were based on the broad definition approach where economic value was placed on a person’s life till the end of his or her life expectancy, not the age of retirement, since the time of death. This approach was supported by recent studies that have criticised the traditional approach – valuing the life years till the age of retirement – as “inaccurate” and “unethical” , 26. NevDirect medical costs were estimated to be US$26,431 per patient in 2013, incurring about US$69 million at the national-level. Of these, chemotherapy-related costs  accounted for more than half of the direct medical costs (60%), followed by inpatient costs (29%) and outpatient costs (12%).Notably, there was no significant difference in direct medical costs as well as direct non-medical costs/morbidity costs between patients who received second-line treatment and those who received BSC following the first-line treatment. Mortality costs could not be estimated by the type of treatment because the costs were estimated based on national mortality statistics . Nevertheless, the previous study , using tFinally, the sizes of direct non-medical costs and morbidity costs appeared to be much smaller than those of direct medical costs and mortality costs. Nevertheless, the impact of these costs should not be neglected; these costs are directly related to the welfare of patients and their caregivers because these are the costs fully borne by them. In addition, these costs would be even greater if intangible costs such as psychological distress and subjective burden experienced by patients and caregivers are fully taken into account.Although this is the first COI study that has estimated the size of AGC burden in Taiwan and can help better prioritise and allocate resources for cancer management, the study suffers from a number of limitations that should be taken into account when interpreting the results. Firstly, this study estimated the burden of AGC based mainly on data from a chart review study previously conducted in Taiwan . While tThis study confirms that AGC exerts a significant economic burden in Taiwan. The total economic burden of AGC was estimated to be about US$423 million in 2013. This represents 0.08% of the Taiwanese economy (GDP). Mortality costs appeared to be the single greatest contributor to the burden, followed by direct medical costs, morbidity costs and direct non-medical costs. Early detection and providing effective treatments may help to reduce its burden on patients, caregivers and society as a whole.


Rice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV) are two closely related fijiviruses transmitted by the small brown planthopper (SBPH) and white-backed planthopper (WBPH), respectively. SRBSDV has a latent period 4 days shorter than that of RBSDV, implying a more efficient spread in insect vector. Currently, the mechanisms underlying this higher efficiency are poorly understood. However, our recent studies have implicated a role of virus induced tubular structures in the dissemination of fijiiruses within their insect vectors.Spodoptera frugiperda (Sf9) cells.Immunofluorescence labeling was performed to visualize and compare the dynamics of P7-1 tubule formation of the RBSDV and SRBSDV in their own vector insects and nonhost Tubule formation of SRBSDV P7-1 was faster than that of RBSDV P7-1. For RBSDV, P7-1 formed tubules were observed at 3-days post-first access to diseased plants (padp) in SBPH. For SRBSDV, these structures were detected as early as 1 day padp in WBPH. Importantly, similar phenomena were observed when P7-1 proteins from the two viruses were expressed alone in Sf9 cells.Our research revealed a relationship between the speed of P7-1 tubule formation and virus dissemination efficiency and also supports a role of such tubular structures in the spread of reoviruses within insect vectors. Many plant viruses are transmitted by insect vectors in a circulative propagative manner . NormallReoviridae is a family of viruses characterized by a non-enveloped icosahedral virion encapsulating a genome composed of 10–12 segments of double stranded RNA [Phytorevirus, Oryzavirus and Fijivirus, respectively, are plant-infecting viruses [nded RNA . The fam viruses –7. All t viruses . In rece viruses –11. In b viruses , 13. In  viruses . Disrupt viruses , 13, 15.Rice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV) are two closely related fijiviruses [iviruses , 18. Howiviruses . Our preiviruses , 14. In 11 viral genome copies/μg rice RNA) for 2 days, and then placed on healthy rice seedlings grown at 26 °C, 16 h light/8 h dark and 70 % relative humidity. The internal organs of 30 insects were dissected at different time points to assess the accumulation and distribution of P7-1 by immunofluorescence microscopy as described previously [To investigate the dynamics of P7-1 tubule formation of RBSDV, 200 s-instar SBPHs were fed on RBSDV-infected rice plants  for 2 days and the dynamic localization of SRBSDV P9-1 and P7-1 was assessed in a similar manner. In contrast to the RBSDV observations, SRBSDV P9-1 and P7-1 antigens were detected as early as 1 day padp in epithelial cells of 51 % WBPH midgut, at which time P7-1 tubular structures were observed  cells in the presence of Cellfectin (Invitrogen) as described previously [The above data suggested a faster tubule formation of SRBSDV than RBSDV. While for RBSDV, tubules were observed one day after the appearance of P7-1 antigens, for SRBSDV, there was no time lag detected between P7-1 accumulation and tubule formation. Interestingly, the faster tubule formation corresponded with the more rapid spread of SRBSDV from the gut to the salivary glands, which, likewise, corresponded with the shorter latent period of this virus in its insect vector. However, because the two viruses use two different insect vectors, there are multiple explanations for this observation. To distinguish among these possibilities and to further clarify the role of P7-1 in this process, we utilized the ability of P7-1 to form tubular structures when expressed alone in a heterologous system . To accoeviously . The celeviously . InteresOverall, our data established a relationship between the speed of P7-1 tubule formation and the spread efficiency of two fijiviruses within their insect vectors. This further confirmed a role of the tubular structures that we have observed across all studies of the viral dissemination of reoviruses within insect vectors , 13, 16.