mteb/CovidQA huggingface dataset:
To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the
peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis.
Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus
inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection.
As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication,
suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.
Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .
Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the
mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.
Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.

Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.
Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively.
HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study.

Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .
To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.

Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) .
Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.
Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral
titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).
Rotavirus A in wild and domestic animals from areas with environmental degradation in the Brazilian Amazon

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298726/

SHA: f3c309c596c20f48f493b77e714ce957d877bdcb

Authors: de Barros, Bruno de Cássio Veloso; Chagas, Elaine Nunes; Bezerra, Luna Wanessa; Ribeiro, Laila Graziela; Duarte Júnior, Jose Wandilson Barboza; Pereira, Diego; da Penha Junior, Edvaldo Tavares; Silva, Julia Rezende; Bezerra, Delana Andreza Melo; Bandeira, Renato Silva; Pinheiro, Helder Henrique Costa; Guerra, Sylvia de Fátima dos Santos; Guimarães, Ricardo José de Paula Souza e; Mascarenhas, Joana D'Arc Pereira
Date: 2018-12-18
DOI: 10.1371/journal.pone.0209005
License: cc-by
Abstract: Acute gastroenteritis is one of the main causes of mortality in humans and young animals. Domestic and mainly wild animals such as bats, small rodents and birds are highly diversified animals in relation to their habitats and ecological niches and are widely distributed geographically in environments of forest fragmentation in some areas of the Amazon, being considered important sources for viruses that affect humans and other animals. Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health. Although the knowledge of the global diversity of enteric viruses is scarce, there are reports demonstrating the detection of rotavirus in domestic animals and animals of productive systems, such as bovines and pigs. The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of
Pará, Brazil, which is characterized as an urbanized area with forest fragments. The fecal specimens were collected from October 2014 to April 2016 and subjected to a Qualitative Real-Time Polymerase Chain Reaction (RT-qPCR), using the NSP3 gene as a target. It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas.
Text: Emerging and reemerging infectious diseases are increasing each year in several countries, with an impact both on human populations and on domestic and wild animals living in areas with considerable forest remnants [1] . Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] .

The populations of wild animals that inhabit forest fragments are strategic groups for studies of public health and the transmission of zoonosis, given that they act as indicators in the assistance and intervention in the human populations, aiming at the prevention of outbreaks and epidemics [3] .
Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] . They represent one of the main causes of mortality in humans, and in young animals, counting for about 25% of mortality [8] . Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] .
The rotavirus (RV) belongs to the Reoviridae family and comprises nine species known as Rotavirus group A to I, with a recent proposal of the J species [13, 14] . Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] . They have a double-stranded ribonucleic acid (dsRNA) genome, divided into 11 segments coding for structural proteins (VP1-VP4, VP6 and VP7) and nonstructural (NSP1-NSP5/NSP6) proteins [16, 17] .
There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] .

Although the results of RVA have already been described globally [12, [23] [24] [25] [26] [27] [28] [29] [30] , in Brazil, the occurrence, diversity and role of rotavirus in these animals are still poorly studied, considering the large number of present species [4, [31] [32] [33] [34] .
In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] .
Therefore, it is important to monitor the health of domestic and wild animals in their natural habitat, especially in areas with anthropic alterations that have an interface with rural communities and enterprises, in order to investigate the occurrence of RVA in this population. These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] .
The qualitative real-time polymerase chain reaction (qRT-PCR) used the NSP3 gene and the TaqMan probe from a highly conserved region of the rotavirus non-structural protein 3 (NSP3), which was previously used in samples from human origin and with low viral loads
Precipitation data were obtained from The Brazilian National Institute of Meteorology (Inmethttp://www.inmet.gov.br/) for the years of capture in the Expedito Ribeiro Settlement (2014) and Açailândia (2015) of the Data Collection Platforms (PCDs) of Belém, located 50 km from Santa Bárbara do Pará, and Tracuateua, located 50 km from Peixe-Boi and 100 km from Viseu. Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field. The municipal boundaries were obtained on the website of the Brazilian Institute of Geography and Statistics (IBGE) (http://www.ibge.gov.br/) and data on deforestation and land use were obtained from the PRODES [43] and TerraClass [44] Projects. PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004.
The satellite image was generated using the sensor Sentinel 2 of the European Space Agency (ESA) (https://sentinel.esa.int/ web/sentinel/user-guides/sentinel-2-msi) with Open Access CC-BY License (http://open.esa.int/) from the years of 2017 and 2018.

All the data obtained was stored in a Geographic Database (BDG). The BDG was imported/ stored in a GIS for the editing of the graphic elements, establishment of topological relations between the elements and their respective attributes, spatial analysis and visualization of the result through thematic maps.

For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use. The selected fragments were distributed within the mesoregions studied, and in each selected fragment fecal samples were randomly collected from domestic and wild animals [45] .
Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed).

The data processing, interpretation, visualization and spatial analysis were performed in ArcGIS software (http://www.arcgis.com/). For the analysis of data related to the determination of the richness, composition and abundance of the fauna of the animals studied in the study area, considering the collection methods adopted and the species available in each city, each sample was considered as an independent sample.

The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] .
For the calculation of the Test T, the Statistica software was used, and the indices of infected animals in the two environments (forest fragment and peridomicile) were calculated for each treatment sampled by collection area, using the software Past 1.92. Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] .

The data obtained for the occurrence of RVA and the questionnaires was inserted into a database for a descriptive analysis of the epidemiological profile of the animal population in the three forest ecosystems studied. In this analysis, descriptive statistical treatments were carried out, using customized "row-columns" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T.
Population study, collection of clinical specimens and laboratory methodology. The flying animals (wild birds and chiroptera) were captured using mist nets which were opened at dawn (4:00 a.m.) and closed in the morning (9:00 a.m.) and were inspected every one hour until the closing, with a sampling effort of 15 days. This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site. The fecal specimens were collected by stimulation of the rectal ampulla with the use of a "Zaragatoa", packed in cryogenic vials, identified, stored in liquid nitrogen, and later sent to the Laboratory.
Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm). In each sample plot, 61 traps were distributed, 20 Shermans and 41 Tomahawks being baited with a mixture made with peanut paste, sardines, cod liver oil and corn meal, as well as fruit like banana, apple and pineapple. All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material. The wild animals were sedated with a combination of ketamine 20mg/kg and xylazine 2mg/kg intramuscularly and subsequently, euthanized with anesthetic overdose of 2% lidocaine in the foramen magnum, according to the recommendation of the National Council for the Control of Animal Experimentation (CONCEA).