A Genomic Portrait of Hepatitis C Virus and MicroRNA-122

Hepatitis C virus (HCV) uniquely requires the liver specific microRNA-122 (miR- 122) for replication, yet global effects on endogenous microRNA (miRNA) targets during infection are unexplored. In this body of work, we employed highthroughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during HCV infection. We demonstrate robust AGO binding on the 5\u27 untranslated region of HCV RNA at known and predicted miR-122 sites, thereby establishing conclusive biochemical evidence of endogenous miR-122 action on HCV RNA that firmly agrees with previous genetic evidence. We further characterize novel AGO binding on HCV RNA to determine its dependence on miR-122, miRNAs generally, replication competence and time. These results establish an unbiased interaction landscape between HCV RNA and cellular miRNAs, mostly miR-122. On the human transcriptome, we observed reduced AGO binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 sponge effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. Based on these results, we describe a quantitative mathematical model of HCV induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets. This in turn may provide an environment fertile for the long-term oncogenic potential of HCV. This last point presented a fitting entree into miR-122 biology, given its known tumor suppressive activity in the liver. To conclude this work, we performed AGO-CLIP in miR-122 knockout mouse livers as well as in human liver samples, to determine the in vivo targetome for this miRNA across two species. Surprisingly, we discovered widespread and non-canonical miR-122 binding throughout the transcriptome. Furthermore, a substantial fraction of this binding was not conserved between mouse and human transcriptomes, despite the fact that miR-122 is highly conserved. These results, in concert with AGOCLIP in HCV infected cells, point to a model where HCV may have evolved the use of miR-122 for its high abundance and its well buffered capacity to be inhibited with minimal detrimental effects to the host, and perhaps benefits for the virus. In sum, this thesis reveals how miR-122 is redistributed in the cell following HCV infection. As a molecular mechanism, chronic inhibition of miR-122 by HCV RNA is proposed to impact, and may very well help induce, the complex constellation of liver diseases that characterize this infection in humans

Impact of Simultaneous Exposure to Arboviruses on Infection and Transmission by Aedes aegypti Mosquitoes

The recent emergence of both chikungunya and Zika viruses in the Americas has significantly expanded their distribution and has thus increased the possibility that individuals may become infected by more than one Aedes aegypti-borne virus at a time. Recent clinical data support an increase in the frequency of coinfection in human patients, raising the likelihood that mosquitoes could be exposed to multiple arboviruses during one feeding episode. The impact of coinfection on the ability of relevant vector species to transmit any of these viruses (that is, their vector competence) has not been determined. Thus, we here expose Ae. aegypti mosquitoes to chikungunya, dengue-2 or Zika viruses, both individually and as double and triple infections. Our results show that these mosquitoes can be infected with and can transmit all combinations of these viruses simultaneously. Importantly, infection, dissemination and transmission rates in mosquitoes are only mildly affected by coinfection

Impact of Extrinsic Incubation Temperature on Natural Selection During Zika Virus Infection of Aedes Aegypti and Aedes Albopictus

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of \u3e 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes

Vector Competence of American Mosquitoes for Three Strains of Zika Virus

In 2015, Zika virus (ZIKV; Flaviviridae; Flavivirus) emerged in the Americas, causing millions of infections in dozens of countries. The rapid spread of the virus and the association with disease outcomes such as Guillain-Barré syndrome and microcephaly make understanding transmission dynamics essential. Currently, there are no reports of vector competence (VC) of American mosquitoes for ZIKV isolates from the Americas. Further, it is not clear whether ZIKV strains from other genetic lineages can be transmitted by American Aedes aegypti populations, and whether the scope of the current epidemic is in part facilitated by viral factors such as enhanced replicative fitness or increased vector competence. Therefore, we characterized replication of three ZIKV strains, one from each of the three phylogenetic clades in several cell lines and assessed their abilities to be transmitted by Ae. aegypti mosquitoes. Additionally, laboratory colonies of different Culex spp. were infected with an American outbreak strain of ZIKV to assess VC. Replication rates were variable and depended on virus strain, cell line and MOI. African strains used in this study outcompeted the American strain in vitro in both mammalian and mosquito cell culture. West and East African strains of ZIKV tested here were more efficiently transmitted by Ae. aegypti from Mexico than was the currently circulating American strain of the Asian lineage. Long-established laboratory colonies of Culex mosquitoes were not efficient ZIKV vectors. These data demonstrate the capacity for additional ZIKV strains to infect and replicate in American Aedes mosquitoes and suggest that neither enhanced virus replicative fitness nor virus adaptation to local vector mosquitoes seems likely to explain the extent and intensity of ZIKV transmission in the Americas

miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence

Hepatitis C virus (HCV) requires the liver specific micro-RNA (miRNA), miR-122, to replicate. This was considered unique among RNA viruses until recent discoveries of HCV-related hepaciviruses prompting the question of a more general miR-122 dependence. Among hepaciviruses, the closest known HCV relative is the equine non-primate hepacivirus (NPHV). Here, we used Argonaute cross-linking immunoprecipitation (AGO-CLIP) to confirm AGO binding to the single predicted miR-122 site in the NPHV 5’UTR in vivo. To study miR-122 requirements in the absence of NPHV-permissive cell culture systems, we generated infectious NPHV/HCV chimeric viruses with the 5’ end of NPHV replacing orthologous HCV sequences. These chimeras were viable even in cells lacking miR-122, although miR-122 presence enhanced virus production. No other miRNAs bound this region. By random mutagenesis, we isolated HCV variants partially dependent on miR-122 as well as robustly replicating NPHV/HCV variants completely independent of any miRNAs. These miRNA independent variants even replicate and produce infectious particles in non-hepatic cells after exogenous delivery of apolipoprotein E (ApoE). Our findings suggest that miR-122 independent HCV and NPHV variants have arisen and been sampled during evolution, yet miR-122 dependence has prevailed. We propose that hepaciviruses may use this mechanism to guarantee liver tropism and exploit the tolerogenic liver environment to avoid clearance and promote chronicity

A Large and Variable Leading Tail of Helium in a Hot Saturn Undergoing Runaway Inflation

Atmospheric escape shapes the fate of exoplanets, with statistical evidence for transformative mass loss imprinted across the mass-radius-insolation distribution. Here we present transit spectroscopy of the highly irradiated, low-gravity, inflated hot Saturn HAT-P-67 b. The Habitable Zone Planet Finder (HPF) spectra show a detection of up to 10% absorption depth of the 10833 Angstrom Helium triplet. The 13.8 hours of on-sky integration time over 39 nights sample the entire planet orbit, uncovering excess Helium absorption preceding the transit by up to 130 planetary radii in a large leading tail. This configuration can be understood as the escaping material overflowing its small Roche lobe and advecting most of the gas into the stellar -- and not planetary -- rest frame, consistent with the Doppler velocity structure seen in the Helium line profiles. The prominent leading tail serves as direct evidence for dayside mass loss with a strong day-/night- side asymmetry. We see some transit-to-transit variability in the line profile, consistent with the interplay of stellar and planetary winds. We employ 1D Parker wind models to estimate the mass loss rate, finding values on the order of $2\times10^{13}$ g/s, with large uncertainties owing to the unknown XUV flux of the F host star. The large mass loss in HAT-P-67 b represents a valuable example of an inflated hot Saturn, a class of planets recently identified to be rare as their atmospheres are predicted to evaporate quickly. We contrast two physical mechanisms for runaway evaporation: Ohmic dissipation and XUV irradiation, slightly favoring the latter.Comment: Submitted to The Astronomical Journa

An ethical framework for global vaccine allocation

In this article, we propose the Fair Priority Model for COVID-19 vaccine distribution, and emphasize three fundamental values we believe should be considered when distributing a COVID-19 vaccine among countries: Benefiting people and limiting harm, prioritizing the disadvantaged, and equal moral concern for all individuals. The Priority Model addresses these values by focusing on mitigating three types of harms caused by COVID-19: death and permanent organ damage, indirect health consequences, such as health care system strain and stress, as well as economic destruction. It proposes proceeding in three phases: the first addresses premature death, the second long-term health issues and economic harms, and the third aims to contain viral transmission fully and restore pre-pandemic activity. To those who may deem an ethical framework irrelevant because of the belief that many countries will pursue "vaccine nationalism," we argue such a framework still has broad relevance. Reasonable national partiality would permit countries to focus on vaccine distribution within their borders up until the rate of transmission is below 1, at which point there would not be sufficient vaccine-preventable harm to justify retaining a vaccine. When a government reaches the limit of national partiality, it should release vaccines for other countries. We also argue against two other recent proposals. Distributing a vaccine proportional to a country's population mistakenly assumes that equality requires treating differently situated countries identically. Prioritizing countries according to the number of front-line health care workers, the proportion of the population over 65, and the number of people with comorbidities within each country may exacerbate disadvantage and end up giving the vaccine in large part to wealthy nations

Human papillomavirus detection in cervical neoplasia attributed to 12 high-risk human papillomavirus genotypes by region

Background: We estimated the proportion of cervical intraepithelial neoplasia (CIN) cases attributed to 14 HPV types, including quadrivalent (qHPV) (6/11/16/18) and 9-valent (9vHPV) (6/11/16/18/31/33/45/52/58) vaccine types, by region Methods: Women ages 15-26 and 24-45 years from 5 regions were enrolled in qHPV vaccine clinical trials. Among 10,706 women (placebo arms), 1539 CIN1, 945 CIN2/3, and 24 adenocarcinoma in situ (AIS) cases were diagnosed by pathology panel consensus. Results: Predominant HPV types were 16/51/52/56 (anogenital infection), 16/39/51/52/56 (CIN1), and 16/31/52/58 (CIN2/3). In regions with largest sample sizes, minimal regional variation was observed in 9vHPV type prevalence in CIN1 (similar to 50%) and CIN2/3 (81-85%). Types 31/33/45/52/58 accounted for 25-30% of CIN1 in Latin America and Europe, but 14-18% in North America and Asia. Types 31/33/45/52/58 accounted for 33-38% of CIN2/3 in Latin America (younger women), Europe, and Asia, but 17-18% of CIN2/3 in Latin America (older women) and North America. Non-vaccine HPV types 35/39/51/56/59 had similar or higher prevalence than qHPV types in CIN1 and were attributed to 2-11% of CIN2/3. Conclusions: The 9vHPV vaccine could potentially prevent the majority of CIN1-3, irrespective of geographic region. Notwithstanding, non-vaccine types 35/39/51/56/59 may still be responsible for some CIN1, and to a lesser extent CIN2/3. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)