bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of the Relative Performance of Pancreas-Specific MicroRNAs in Rat Plasma as Biomarkers of Pancreas Injury
Authors: Erdos Zoltan , Barnum John E , Wang Erjia , DeMaula Christopher , Dey Paritosh Markus , Forest Thomas , Bailey Wendy J , Glaab Warren E 
Published: August 2019

AbstractDrug-induced pancreatic injury (DIPI) has become linked in recent years to many commonly prescribed medications from several pharmacological classes. Diagnosis is currently most often focused on identification of acute pancreatitis and generally based on subjective clinical assessment and serum amylase and lipase enzymatic activity, which have been criticized as being insufficiently sensitive and specific. The lack of novel noninvasive biomarkers of DIPI can impede the advancement of drug candidates through nonclinical development and translation into clinical settings. Pancreas-specific microRNAs (miRNAs) are currently being evaluated as biomarkers of DIPI that may outperform and/or add value to the interpretation of amylase and lipase. To assess the relative performance of these novel miRNAs, a comprehensive evaluation was conducted to determine the sensitivity and specificity of detecting DIPI in rats. Four miRNAs were evaluated (miR-216a-5p, miR-216b-5p, miR-217-5p, and miR-375-3p) in plasma from 10 studies in which rats were treated with known pancreatic toxicants to assess sensitivity, and from 10 different studies in which toxicity was evident in tissues other than pancreas to assess specificity. The candidate miRNA biomarker performance was compared with amylase and lipase, and receiver operator characteristics (ROC) were determined. Analysis of ROCs demonstrated that all four miRNAs outperformed amylase and lipase in monitoring acute pancreatic injury defined as acinar cell degeneration/necrosis. Specifically, miR-217-5p had the highest performance among all biomarkers assessed. The increased sensitivity and specificity of these miRNAs support their use as biomarkers of DIPI, thereby adding value to the interpretation of amylase and lipase measurements in nonclinical studies. The potential for miRNAs to serve as translational biomarkers in the clinic for the monitoring of DIPI is also supported by this investigation.
A1 The role of virus-antibody co-evolution in the development of a V3-glycan-directed HIV-1 broadly neutralizing antibody lineage
Authors: Kitchin D , Bhiman J , Mvududu D , Oosthuysen B , Lambson B , Madzorera S , Anthony C , Abdool Karim S S , Garrett N J , Doria-Rose N A , Mascola J R , Morris L , Moore P L 
Published: August 2019

Abstract
Broadly neutralizing antibodies (bNAbs) are essential for a preventative HIV-1 vaccine but have not been elicited through vaccination. bNAbs develop in 20–30 per cent of HIV-1 infections and often target the V3-glycan epitope of the HIV envelope protein (Env). In these individuals, virus-antibody co-evolution is thought to drive the maturation of antibody lineages to neutralization breadth. We used deep sequencing of env genes and antibody transcripts from fourteen time points spanning the first 3 years of infection to characterize the virus-antibody co-evolution in donor CAP255 who developed V3-glycan-specific bNAbs. Sequencing and cloning of env genes, followed by neutralization assays, were used to identify Env mutations associated with neutralization escape from two bNAbs (CAP255.G3 and CAP255.C5) isolated at 149 weeks post-infection (wpi). Sequencing data indicated that CAP255 was co-infected by three related viral variants, all of which had an intact N332 glycan, which persisted in > 90 per cent of later viruses. A recombinant V3-region became fixed from 8 wpi, conferring slight neutralization resistance to CAP255.G3/C5 and other V3-glycan bNAbs. Later, T415R/K substitutions in V4 emerged by 51 wpi and were associated with complete viral escape from CAP255.G3/C5, though not from the polyclonal plasma response. All 93-week and later Envs were resistant to CAP255.G3/C5 and V3-glycan bNAb PGT135. Viral escape by 51 wpi suggested that the CAP255 bNAbs arose earlier, driving escape, but persisted to 149 weeks. This was supported by preliminary deep sequencing of the antibody repertoire that indicated bNAb lineage members were already present in the plasma at 39 wpi. Escape from V3-glycan bNAbs via T415R/K mutations have not previously been shown, suggesting a novel mode of recognition within the V3-glycan supersite. Further work will focus on identifying the bNAb-initiating Env and intermediate bNAb lineage members that were capable of engaging contemporaneous Env neutralization escape mutants. Characterization of Envs that engaged bNAb precursors, as well as those that selected for broader members of the bNAb lineage, will inform the design of immunogens capable of eliciting V3-glycan bNAbs in a novel HIV-1 vaccine regimen.
A5 Near full-length HIV-1 genome sequencing in newly diagnosed individuals in Sweden
Authors: Andersson E , Aralaguppe S , Thebo L , Sönnerborg A , Neogi U , Albert J 
Published: August 2019

Abstract
The Swedish HIV-1 epidemic is characterized by a high diversity in HIV subtypes and recombinants as a result of migration. To study the time from infection through viral diversification, transmission patterns, and drug resistance in minor quasispecies, a robust protocol for pan-genotypic near full-length HIV-1 genome (HIV-NFLG) next-generation sequencing (NGS) is key. Our group has established two protocols for HIV-NFLG on the Illumina platform that we aim to compare and, if necessary, modify to find a method with optimized coverage, depth, and subtype inclusivity. Zanini et al. (https://doi.org/10.7554/eLife.11282.001) have developed a method with one-step RT-PCR with six overlapping primer sets, followed by NGS and quality filtering and assembly with in-house methods. Aralaguppe et al. (https://doi.org/10.1016/j.jviromet.2016.07.010) have designed amplification in two fragments, followed by multiplexed NGS and quality control and assembly with Iterative Virus Assembler and VICUNA. Both methods have high coverage per nucleotide and low error rates in amplification and sequencing and can reliably identify SNPs at 1 per cent of the viral population with linkage within the quasispecies. Subtype inclusivity remains a challenge even though both methods show success in amplifying and sequencing subtypes B, C, and the common recombinants 01_AE and 02_AG. Therefore, we aim to evaluate and optimize our NFLG NGS methods on a panel of patient samples that more completely reflects HIV-1 diversity in Sweden. Patient samples from fifty treatment-naïve viremic individuals representing the genotypic HIV-1 panorama in Sweden, including CRFs, are being amplified and sequenced by both protocols. Coverage of the genome, error rate, and possible depth of quasispecies analysis is being evaluated. We will compare number of reads, coverage across the HIV genome, and representation of minor single nucleotide variants as well as subtype inclusivity and impact of plasma RNA levels. To do this we will use an in-house bioinformatic pipeline. The NFLG sequences will also be analyzed with phylogenetic tools for determination of subtypes including CRFs and URFs.
A9 A method to obtain full-length HIV proviral sequences and their sites of integration
Authors: Patro Sean C , Wu Xiaolin , Guo Shuang , Bale Michael J , Wiegand Ann , Spindler Jonathan , Fennessey Christine M , Halvas Elias K , Keele Brandon F , Hughes Stephen H , Mellors John W , Coffin John M , Rausch Jason W , Kearney Mary F 
Published: August 2019

Abstract
Accurate definition of the HIV-1 reservoir on antiretroviral therapy (ART) is of paramount importance to the development of curative strategies. Much of this reservoir is derived from clonal expansion of latently infected CD4+ T cells. Methods used to characterize the reservoir include near full-length single-genome sequencing (NFL-SGS) and integration site analysis (ISA). However, current technologies do not link the intact proviruses detected by NFL-SGS to their sites of integration. Therefore, we developed a method to obtain both near full-length single-proviral sequences and their sites of integration. We call our method full-length integrated proviral single-genome sequencing (FLIP-SGS). Genomic DNA from ACH2 and CEM cells mixed at 1:1,000, or patient samples were diluted to a single proviral endpoint. An in-house, optimized whole genome amplification (WGA) method was performed on wells at the endpoint, generating multiple copies of all DNA molecules within each well. The number of proviral copies after WGA was determined by droplet digital PCR targeting the long terminal region (LTR). Forty per cent of each WGA reaction was used to obtain the provirus–host integration sites with ISA (linker ligation, nested PCR, and Illumina sequencing). The remaining fraction was used to amplify the full-length proviruses in four overlapping fragments (LTR-pol, gag-int, int-env, and env-LTR) for Sanger sequencing. WGA performed on the endpoint-diluted ACH2:CEM DNA amplified single-copy HIV-1 proviral templates greater than 500-fold, making it possible to obtain unique integration sites from single proviruses in ACH2 cells, including one that was previously reported (in the NT5C3A gene on chromosome 7) and two that were not previously reported (in the EIF4ENIF1 gene of chromosome 22 and an unknown region of chromosome 6). Near full-length PCR amplification and Sanger sequencing was performed on proviruses integrated in the NT5C3A gene. FLIP-SGS was applied to peripheral blood mononuclear cells from one HIV-1 infected donor with viremia suppressed on ART and yielded integration sites of four genomes that appear to contain large internal deletions. We report a method for near full-length HIV-1 single-genome sequencing combined with host integration site detection that we call FLIP-SGS. This assay will further define clonal expansion of infected CD4+ T cells as a mechanism that maintains the HIV-1 reservoir and as the source of identical sequences observed during therapy and rebound, rather than from ongoing replication.
A16 Next-generation sequencing to detect transmitted drug resistance mutations in Romanian people who inject drugs
Authors: Surleac Marius , Paraschiv Simona , Nicolae Ionelia , Banica Leontina , Vlaicu Ovidiu , Jipa Raluca , Abagiu Adrian , Otelea Dan 
Published: August 2019

Abstract
Romania has faced an HIV outbreak among people who inject drugs (PWID) since 2011. The introduction of so-called ‘legal highs’ (amphetamine-type stimulants) on the drug market a few years prior contributed substantially to this outbreak. Next-generation sequencing (NGS) provides the possibility to detect drug resistance mutations with higher sensitivity than Sanger sequencing. The aim of this study was to search for transmitted drug resistance (TDR) mutations in strains from PWID recently diagnosed with HIV infection by parallel use of Sanger sequencing and NGS. The study was conducted on strains from 34 PWID diagnosed with HIV infection between 2016 and 2017. Sequencing was performed for the pol (PR, RT, and INT) and env (V2-V3 loop) regions. Sanger sequencing was performed with the commercial ViroseqTMHIV-1 Genotyping system (Abbott Laboratories) and with an in-house protocol for the env gene. NGS was performed in the same genomic regions using Nextera DNA Library Preparation Kit (Illumina) and the Miseq instrument (Illumina). NGS data were processed for error correction, read mapping, and detection of drug resistance mutations with HIV-1 Deepchek analysis software. Geno2pheno algorithm was used for viral tropism prediction and the WHO 2009 list for TDRM analysis. By using NGS, we detected seven cases (20.6%) of TDR in PWID and only two cases (5.8%) with standard sequencing. The TDR mutations detected by NGS were K103N, K101EN, Y181C, T215S in RT gene, I54V and M46L in PR, and none in INT. Two NNRTI mutations (K103N and K101EN) were detected in the same sample. Most of the TDR identified were present in the minority population (between 1% and 2% of the total reads) explaining the higher sensitivity of NGS method compared with standard sequencing. No significant differences were observed between these two methods when tropism prediction was analyzed. The majority of the viruses circulating in this group were R5-tropic. All strains showed more resistance mutations when analyzed by deep sequencing than by Sanger sequencing and more than previously observed in other risk groups. NGS proved to be a sensitive tool to detect TDR in newly infected PWID.
A28 Spatial spread of highly pathogenic avian influenza A (H5N8) virus in Italy, 2017–8
Authors: Zecchin B , Fusaro A , Zamperin G , Milani A , Schivo A , Salomoni A , Salviato A , Ormelli S , Marciano S , Terregino C , Monne I 
Published: August 2019

Abstract
In winter 2016–7 the highly pathogenic avian influenza (HPAI) virus, H5N8 subtype, clade 2.3.4.4 group B, circulated extensively both in wild and domestic birds in Europe. Northern Italy was hit by three epidemic waves: the first in January–May 2017, the second in July–December 2017, and the latest in March 2018. To genetically characterize the viruses circulating in Italy we used the Illumina MiSeq platform to sequence the complete genome of representative viruses from each infected farm, for a total of 86 cases in poultry and 17 in wild birds. Maximum likelihood phylogenetic analyses performed using PhyML version 3.1 identified multiple viral introductions of distinct genotypes of HPAI H5N8 viruses in Italy at the beginning of the epidemic (January–February 2017). During the second epidemic wave a single genetic group originating from the virus A/wild duck/Poland/82A/2016 seemed to have been selected, further evolving into two different clusters, namely Italy-A and Italy-B. We identified four clusters of secondary outbreaks, the largest being the epidemic in the province of Brescia between October 2017 and March 2018, which had affected 26 farms. Evolutionary and phylogeographic analyses performed with the BEAST v1.8.4 package (applying a Bayesian Markov chain Monte Carlo approach, using a constant size coalescent tree prior and a SRD06 model of nucleotide substitution) indicated that different viral introductions had probably occurred through migratory birds from West Russia, Siberia, Central and East Europe. The discrete and continuous phylogeographic analyses showed that group Italy-A had probably emerged between February and April 2017 in the province of Mantua and had then spread eastwards, circulating in the Veneto region and eastern Lombardy; on the contrary, Italy-B had arisen between March and July 2017 in the central part of Lombardy and had spread westwards, circulating in the western part of Lombardy, Emilia Romagna, and Piedmont regions. This study was instrumental to reconstruct the virus dissemination routes and indicated that wild and domestic birds from Lombardy most likely represented the key source for the re-emergence and spread of the HPAI virus during the second and the third epidemic waves. This key spatial information will help to define appropriate disease control strategies.
A31 Diversity change of influenza A (H3N2) strains circulating in Brazil during 2017–8: What to expect in the coming winter?
Authors: Resende P C , Caetano B , Matos A , Lopes J , Garcia C , Miranda M , Born P , Motta F C , Brown D , Siqueira M 
Published: August 2019

Abstract
The H3N2 subtype of influenza A (H3N2) was the predominant strain during the early months of the 2017 influenza epidemic in Brazil. In Australia, it was responsible for a strong and prolonged 2017 season and reached the Northern hemisphere causing an intense 2017/8 influenza season. Several genetic and antigenic A(H3N2) variants were circulating, which made the decision about which strain to incorporate into the influenza vaccine challenging. For 2018, the WHO selected a new H3N2 strain, A/Singapore/INFIMH-16-0019/2016-like, to replace the strain A/HongKong/4801/2014-like in the Southern Hemisphere trivalent vaccine. The aim of this study was to describe the genetic diversity of influenza A (H3N2) viruses circulating in Brazil between January 2017 and January 2018, checking the match between the vaccines and worldwide circulating strains with the Brazilian influenza strains. Hemagglutinin gene sequencing of the influenza A (H3N2) was performed, followed by a phylogenetic reconstruction using additional database sequences to define genetic groups and compare with other worldwide circulating strains. We observed a large diversity of H3N2 genetic clusters, including 3C.2a, 3C.2a1, 3C.3a, and their subgroups. During the 2016–7, inter-epidemic and 2017 epidemic period the cluster most frequently detected belonged to clade 3C.2a1 (148/185; 80.0%), a distinct group related to the 2017 vaccine strain A/HongKong/4801/2014-like (3C.2a). However, the genetic profile changed during the study period and in the inter-epidemic season 2017–8 the most commonly detected genetic group was the 3C.2a cluster (43/58; 74.1%). Inside this cluster, the majority (34/43; 79.1%) of strains belonged to a single genetic 3C.2a subgroup 2 (3C.2a2), bearing antigenic substitutions T131K and R142K (site A) and R261Q (site E). The dominance of this 3C.2a2 in the 2017–8 inter-epidemic period in Brazil was similar to the 2017–8 season in Europe and Canada according their surveillance data. The new vaccine strain has five to six antigenic changes in comparison to the predominant 3C.2a2 circulating in South America since September 2017 until now. It is possible that the vaccine mismatch will not protect the population against a majority of circulating strains. Surveillance of the vaccine effectiveness supported by antigenic and serological analysis are necessary to prove this hypothesis. However, this highlights the difficulty of vaccine strain selection and highlights the need for of a universal influenza vaccine.
A32 Genomic surveillance of Zika virus transmission in the Amazonas State, Brazil
Authors: Giovanetti Marta , de Jesus Jaqueline Goes , Joshua Quick , Claro Ingra Morales , Junior Joilson Xavier , Hill Sarah C , Renato Souza , Paz Paola , Lourenco Jose , Dellicour Simon , Sabino Esther , Loman Nicholas J , Naveca Felipe , Pybus Oliver G , Faria Nuno Rodrigues , Carlos Junior Alcantara Luiz 
Published: August 2019

Abstract
Zika virus (ZIKV) has caused an unprecedented epidemic linked to severe congenital syndromes. Transmission of ZIKV in the Americas was first confirmed in May 2015 in northeast Brazil, though the virus was likely introduced 1–2 years prior to its detection. Manaus, the capital city of the Amazonas State, the largest territory of any state in Brazil and the main economic center in the northern region, reported between 2016 and 2017 more than 2,327 suspected cases of ZIKV infection. To gain insights into the timing, source, and likely route(s) of ZIKV introduction in the Amazonas State, we tracked the virus by sequencing ZIKV genomes from infected patients. Using nanopore sequencing technology, we generated 56 Brazilian ZIKV genomes from Manaus city in the Amazonas state, sampled from human cases. On the basis of available sequences of isolates from the Americas, the Manaus sequences, we analyzed fell within a single strongly supported monophyletic clade (bootstrap support = 99%, posterior support = 1.00) that belongs to the Asian genotype. Genetic analysis suggests the outbreak most likely originated from transmission cycles not previously identified in North Brazil and not from a separate introduction into the Americas. Molecular dating analysis indicates that the outbreak was caused by a single founder strain that is estimated to have arrived in Manaus around February 2015. By analyzing surveillance and genetic data, we discovered that ZIKV moved among transmission zones in Manaus. Geographical analysis further indicates that the Northern part of the Manaus regions has a high transmission potential for ZIKV. Our work illustrates that near-real time genomics in the field can augment traditional approaches to infectious disease surveillance and control. Estimated dates for the international spread of ZIKV from the north region indicate the persistence of the virus transmission in recipient regions. Our study provides an understanding of how ZIKV initiates transmission in new regions.
A37 Transmission success of dengue virus type 1 lineages in a dynamic virus population: An evolutionary view
Authors: Koo Carmen , Xu Helen , Rajarethinam Jayanthi , Lai Yee Ling , Ng Lee-Ching , Hapuarachchi Hapuarachchige Chanditha 
Published: August 2019

Abstract
Arbovirus transmission involves an interplay between host, virus, and environmental factors. Because of the complexity of interactions, the transmission success of arboviruses could either be a function of viral fitness or be stochastic. In the present study, using 1,963 envelope (E) gene sequences and 239 whole genomes, we conducted a large-scale molecular epidemiological analysis of a dengue virus type 1 (DENV-1) population to understand the transmission success, evolution, and dispersal patterns of different lineages of DENV-1 circulating in Singapore from 2011 to 2016. The study population was highly dynamic and heterogeneous. However, only a handful of genetically distinct strains (n = 6) established sustained transmission, but at variable levels of dominance. Phylogeographic analysis revealed a weak spatial clustering and 35 well-supported diffusion pathways, implying widespread and complex dispersal of these strains in local settings. Yet, the dominant strains were neither evolving faster than less dominant ones nor under positive selection. These observations suggested that lineage dominance was likely to be stochastic and opportunistically driven by non-viral factors such as host immune pressure and vector abundance. Our findings, therefore, emphasize the implications of understanding the vector and human factors in parallel to virus dynamics on continuing efforts to control the arbovirus disease transmission in endemic regions.