Biology subjects

Maljkovic Berry, I.

Publications and source records attributed to Maljkovic Berry, I..

11 recordsLinked to original sources

bioRxiv2022

Intrahost SARS-CoV-2 k-mer identification method (iSKIM) for rapid detection of mutations of concern reveals emergence of global mutation patterns

Despite unprecedented global sequencing and surveillance of SARS-CoV-2, timely identification of the emergence and spread of novel variants of concern (VoCs) remains a challenge. Several million raw genome sequencing runs are now publicly available. We sought to survey these datasets for intrahost variation to study emerging mutations of concern. We developed iSKIM ("intrahost SARS-CoV-2 k-mer identification method") to relatively quickly and efficiently screen the many SARS-CoV-2 datasets to identify intrahost mutations belonging to lineages of concern. Certain mutations surged in frequency as intrahost minor variants just prior to, or while lineages of concern arose. The Spike N501Y change common to several VoCs was found as a minor variant in 834 samples as early as October 2020. This coincides with the timing of the first detected samples with this mutation in the Alpha/B.1.1.7 and Beta/B.1.351 lineages. Using iSKIM, we also found that Spike L452R was detected as an intrahost minor variant as early as September 2020, prior to the observed rise of the Epsilon/B.1.429/B.1.427 lineages in late 2020. iSKIM rapidly screens for mutations of interest in raw data, prior to genome assembly, and can be used to detect increases in intrahost variants, potentially providing an early indication of novel variant spread.

genomics↗

bioRxiv2021

High confidence identification of intra-host single nucleotide variants for person-to-person influenza transmission tracking in congregate settings

Influenza within-host viral populations are the source of all global influenza diversity and play an important role in driving the evolution and escape of the influenza virus from human immune responses, antiviral treatment, and vaccines, and have been used in precision tracking of influenza transmission chains. Next Generation Sequencing (NGS) has greatly improved our ability to study these populations, however, major challenges remain, such as accurate identification of intra-host single nucleotide variants (iSNVs) that represent within-host viral diversity of influenza virus. In order to investigate the sources and the frequency of called iSNVs in influenza samples, we used a set of longitudinal influenza patient samples collected within a University of Maryland (UMD) cohort of college students in a living learning community. Our results indicate that technical replicates aid in removal of random RT-PCR, PCR, and platform sequencing errors, while the use of clonal plasmids for removal of systematic errors is more important in samples of low RNA abundance. We show that the choice of reference for read mapping affects the frequency of called iSNVs, with the sample self-reference resulting in the lowest amount of iSNV noise. The importance of variant caller choice is also highlighted in our study, as we observe differential sensitivity of variant callers to the mapping reference choice, as well as the poor overlap of their called iSNVs. Based on this, we develop an approach for identification of highly probable iSNVs by removal of sequencing and bioinformatics algorithm-associated errors, which we implement in phylogenetic analyses of the UMD samples for a greater resolution of transmission links. In addition to identifying closely related transmission connections supported by the presence of highly confident shared iSNVs between patients, our results also indicate that the rate of minor variant turnover within a host may be a limiting factor for utilization of iSNVs to determine patient epidemiological links.

genomics↗

bioRxiv2021

Identification of Japanese Encephalitis Virus Genotype V and Other Mosquito-borne Viruses in Camp Humphreys, Republic of Korea, using Metagenomic Analysis

Recent outbreaks of emerging and re-emerging viruses such as Zika, West Nile and Japanese encephalitis (JEV) viruses have shown that timely detection of novel arboviruses with epidemic potential is essential to mitigate human health risks. There have been rising concerns that an emergent JEV genotype (genotype V, GV) is circulating in Asia, against which the current US-FDA-approved JEV vaccine may not be efficacious. To ascertain if JEV GV and other arboviruses are circulating in East Asia, we conducted next-generation sequencing on 260 pools of Culex tritaeniorhynchus and Culex bitaeniorhynchus mosquitoes (6,540 specimens) collected at Camp Humphreys, Republic of Korea (ROK), from mid-May - October 2018. Metagenomic analysis demonstrated a highly abundant and diverse virome with correlates of health and ecological relevance. Additionally, two complete JEV GV genome sequences were obtained from separate mosquito pools, indicating that JEV GV is circulating in the Pyeongtaek area near Seoul, ROK. Retrospective sample and sequence analyses showed that JEV GV was also present in 2016 mosquito pools collected in Seoul, ROK. Sequence-based analysis of JEV GV indicates a divergent genotype that is the most distant from the GIII derived live attenuated SA14-14-2 vaccine strain. A GV E protein investigation and 3D modeling in context to SA14-14-2 indicated likely regions responsible for reduced antibody affinity, including clusters of significant amino acid changes at externally exposed domains. These data highlight the critical need for continued mosquito surveillance as a means of detecting and identifying emerging and re-emerging arboviruses of public health relevance. Importantly, our results emphasize recent concerns that there may be a possible shift in the circulating JEV genotype in East Asia and highlights the critical need for a vaccine proven to be efficacious against this re-emergent virus.

genomics↗

bioRxiv2021

A comparative recombination analysis of human coronaviruses and implications for the SARS-CoV-2 pandemic

The SARS-CoV-2 pandemic prompts evaluation of recombination in human coronavirus (hCoV) evolution. We undertook recombination analyses of 158,118 public seasonal hCoV, SARS-CoV-1, SARS-CoV-2 and MERS-CoV genome sequences using the RDP4 software. We found moderate evidence for 8 SARS-CoV-2 recombination events, two of which involved the spike gene, and low evidence for one SARS-CoV-1 recombination event. Within MERS-CoV, 229E, OC43, NL63 and HKU1 datasets, we noted 7, 1, 9, 14, and 1 high-confidence recombination events, respectively. There was propensity for recombination breakpoints in structural genes, and recombination severely skewed the temporal structure of these data, especially for NL63 and OC43. Bayesian time-scaled analyses on recombinant-free data indicated the sampled diversity of seasonal CoVs emerged in the last 70 years, with 229E displaying continuous lineage replacements. These findings emphasize the importance of genomic based surveillance to detect recombination in SARS-CoV-2, particularly if recombination may lead to immune evasion.

genomics↗

bioRxiv2020

Rapid High Throughput Whole Genome Sequencing of SARS-CoV-2 by using One-step RT-PCR Amplification with Integrated Microfluidic System and Next-Gen Sequencing

The long-lasting global COVID-19 pandemic demands timely genomic investigation of SARS-CoV-2 viruses. Here we report a simple and efficient workflow for whole genome sequencing utilizing one-step RT-PCR amplification on a microfluidic platform, followed by MiSeq amplicon sequencing. The method uses Fluidigm IFC and instruments to amplify 48 samples with 39 pairs of primers in a single step. Application of this method on RNA samples from both viral isolate and clinical specimens demonstrate robustness and efficiency of this method in obtaining the full genome sequence of SARS-CoV-2.

molecular biology↗

bioRxiv2020

Vector and cell-culture passaging of dengue clinical samples for virus isolation and amplification does not significantly change genome consensus or frequencies of intra-host viral variants

Intra-host single nucleotide variants (iSNVs) have been increasingly used in genomic epidemiology to increase phylogenetic resolution and reconstruct fine-scale outbreak dynamics. These analyses are preferably done on sequence data from direct clinical samples, but in many cases due to low viral loads, there might not be enough genetic material for deep sequencing and iSNV determination. Isolation of the virus from clinical samples with low passage number increases viral load, but to date, no studies have investigated how dengue virus (DENV) culture isolation from a clinical sample impacts the consensus sequence, and there is no information on the intra-host virus population changes that may result from viral isolation. In this study, we investigate consensus and iSNV frequency differences between DENV sequenced directly from clinical samples and their corresponding low-passage isolates. Twenty five DENV1 and DENV2 positive sera and their corresponding viral isolates (T.splendens inoculation and C6/36 passage) were obtained from a prospective cohort study in the Philippines. These were sequenced on MiSeq with minimum nucleotide depth of coverage of 1000x, and iSNVs were detected using LoFreq. For both DENV1 and DENV2, we found that the nucleotide call concordance (including called iSNVs with variant cutoff at 5%) between direct sera sample and its cultured virus was on average 99.99%. There were a maximum of one consensus nucleotide difference between clinical sample and isolate. Interestingly, we found that iSNV frequencies were also largely preserved between the samples, with an average difference in minor variant frequency of 6.8% (95CI: 3.6%-10%) and 9.6% (95CI: 7%-12.2%) for DENV1 and DENV2, respectively. Furthermore, we found no significant differences in either DENV1 or DENV2 between the sample pairs (clinical sample and isolate) in their number of iSNV positions per genome, or in the difference in variant frequencies (p=0.36 and p=0.13, respectively, F-test). Our results show that low-passage DENV isolates may be used for identification of the majority of their human-derived within-host variant populations, which are increasingly being used for precision tracking of DENV and other RNA viruses.

evolutionary biology↗

bioRxiv2020

Hidden genomic diversity of SARS-CoV-2: implications for qRT-PCR diagnostics and transmission

The COVID-19 pandemic has sparked an urgent need to uncover the underlying biology of this devastating disease. Though RNA viruses mutate more rapidly than DNA viruses, there are a relatively small number of single nucleotide polymorphisms (SNPs) that differentiate the main SARS-CoV-2 clades that have spread throughout the world. In this study, we investigated over 7,000 SARS-CoV-2 datasets to unveil both intrahost and interhost diversity. Our intrahost and interhost diversity analyses yielded three major observations. First, the mutational profile of SARS-CoV-2 highlights iSNV and SNP similarity, albeit with high variability in C>T changes. Second, iSNV and SNP patterns in SARS-CoV-2 are more similar to MERS-CoV than SARS-CoV-1. Third, a significant fraction of small indels fuel the genetic diversity of SARS-CoV-2. Altogether, our findings provide insight into SARS-CoV-2 genomic diversity, inform the design of detection tests, and highlight the potential of iSNVs for tracking the transmission of SARS-CoV-2.

genomics↗

bioRxiv2020

Analysis of cell-associated DENV RNA by oligo(dT) primed 5' capture scRNAseq

Dengue is one of the most widespread vector-borne viral diseases in the world. However, the size, heterogeneity, and temporal dynamics of the cell-associated viral reservoir during acute dengue virus (DENV) infection remains unclear. In this study, we analyzed cells infected in vitro with DENV and PBMC from an individual experiencing a natural DENV infection utilizing 5 capture single cell RNA sequencing (scRNAseq). Both positive- and negative-sense DENV RNA was detected in reactions containing either an oligo(dT) primer alone, or in reactions supplemented with a DENV-specific primer. The addition of a DENV-specific primer did not increase the total amount of DENV RNA captured or the fraction of cells identified as containing DENV RNA. However, inclusion of a DENV-specific cDNA primer did increase the viral genome coverage immediately 5 to the primer binding site. Furthermore, while the majority of intracellular DENV sequence captured in this analysis mapped to the 5 end of the viral genome, distinct patterns of enhanced coverage within the DENV polyprotein coding region were observed. The 5 capture scRNAseq analysis of PBMC not only recapitulated previously published reports by detecting virally infected memory and naive B cells, but also identified cell-associated genomic variants not observed in contemporaneous serum samples. These results demonstrate that oligo(dT) primed 5 capture scRNAseq can detect DENV RNA and quantify virus-infected cells in physiologically relevant conditions, and provides insight into viral sequence variability within infected cells. IMPORTANCEDengue is one of the most widespread vector-borne viral diseases in the world. However, it is still unclear which cells harbor virus during DENV infection, and how viral reservoirs in serum and infected cells are related. These results demonstrate for the first time that intracellular DENV RNA can be identified and infected cells quantified by 5 capture scRNAseq. This strategy provides a significantly greater throughput and higher sensitivity than previously published methods and has the potential to provide additional information on the genomic heterogeneity of intracellular DENV RNA.

microbiology↗

bioRxiv2019

The evolution of dengue-2 viruses in Malindi, Kenya and greater East Africa: epidemiological and immunological implications

Kenya experiences a substantial burden of dengue, yet there are very few DENV-2 sequence data available from this country and indeed the entire continent of Africa. We therefore undertook whole genome sequencing and evolutionary analysis of fourteen dengue virus (DENV)-2 strains sampled from Malindi sub-County Hospital during the 2017 DENV-2 outbreak in the Kenyan coast. We further performed an extended East African phylogenetic analysis, which leveraged 26 complete African env genes. Maximum likelihood analysis showed that the 2017 outbreak was due to the Cosmopolitan genotype, indicating that this has been the only confirmed human DENV-2 genotype circulating in Africa to date. Phylogeographic analyses indicated transmission of DENV-2 viruses between East Africa and South/South-West Asia. Time-scaled genealogies show that DENV-2 viruses are spatially structured within Kenya, with a time-to-most-common-recent ancestor analysis indicating that these DENV-2 strains were circulating for up to 5.38 years in Kenya before detection in the 2017 Malindi outbreak. Selection pressure analyses indicated sampled Kenyan DENV strains uniquely being under positive selection at 6 sites, predominantly across the non-structural genes, and epitope prediction analyses showed that one of these sites corresponds to a putative predicted MHC-I CD8+ DENV-2 Cosmopolitan virus epitope only evident in a sampled Kenyan virus. Taken together, our findings indicate that the 2017 Malindi DENV-2 outbreak arose from a strain which had circulated for several years in Kenya before recent detection, has experienced diversifying selection pressure, and may contain new putative immunogens relevant to vaccine design. These findings prompt further genomic epidemiology studies in this and other Kenyan locations to further elucidate the transmission dynamics of DENV in this region. Author summary (non-technical)Kenya experiences a substantial burden of dengue, yet the patterns of dengue spread in this region are unclear. Evolutionary analyses of dengue virus strain sequences could offer major insights into the spread of dengue viruses in this region, but there are very few DENV-2 sequence data available from this country and indeed the entire continent of Africa. We therefore undertook whole genome sequencing and evolutionary analysis of fourteen dengue virus (DENV)-2 strains sampled from Malindi sub-County Hospital during the 2017 DENV-2 outbreak in the Kenyan coast. We further performed an extended East African phylogenetic analysis, which leveraged 26 complete African env genes. Our results indicated transmission of DENV-2 viruses between East Africa and South/South-West Asia, that there is localized spread of DENV-2 viruses within Kenya, and that Kenyan DENV-2 strains were circulating for up to 5.38 years in Kenya before detection in the 2017 Coastal outbreak. We further performed selection pressure analyses and identified possible new markers of DENV-2 immune recognition specific to this population, with relevance to vaccine design.

genomics↗

bioRxiv2018

Recent outbreaks of chikungunya virus (CHIKV) in Africa and Asia are driven by a variant carrying mutations associated with increased fitness for Aedes aegypti

BackgroundIn 2016, a chikungunya virus (CHIKV) outbreak was reported in Mandera, Kenya. This was the first major CHIKV outbreak in the country since the global re-emergence of this virus, which arose as an initial outbreak in Kenya in 2004. Therefore, we collected samples and sequenced viral genomes from the 2016 Mandera outbreak.\n\nMethodology/Principal FindingsAll Kenyan genomes contained two mutations, E1:K211E and E2:V264A, recently reported to have an association with increased infectivity, dissemination and transmission in the Aedes aegypti (Ae. aegypti) vector. Phylogeographic inference of temporal and spatial virus relationships using Bayesian approaches showed that this Ae. aegypti adapted strain emerged within the East, Central, and South African (ECSA) lineage of CHIKV between 2005 and 2008, most probably in India. It was also in India where the first large outbreak caused by this strain appeared, in New Delhi, 2010. More importantly, our results also showed that this strain is no longer contained to India, and that it has more recently caused several major outbreaks of CHIKV, including the 2016 outbreaks in India, Pakistan and Kenya, and the 2017 outbreak in Bangladesh. In addition to its capability to cause large outbreaks in different regions of the world, this CHIKV strain has the capacity to replace less adapted wild type strains in Ae. aegypti-rich regions. Indeed, all the latest full CHIKV genomes of the ECSA Indian Ocean Lineage (IOL), from the regions of high Ae. aegypti prevalence, carry these two mutations, including samples collected in Japan, Australia, and China.\n\nConclusions/SignificanceOur results point to the importance of continued genomic-based surveillance of this strains global spread, and they prompt urgent vector competence studies in Asian and African countries, in order to assess the level of vector receptiveness, virus transmission, and the impact this might have on this strains ability to cause major outbreaks.\n\nAuthor summaryChikungunya virus (CHIKV) causes a debilitating infection with high fever, intense muscle and bone pain, rash, nausea, vomiting and headaches, and persistent and/or recurrent joint pains for months or years after contracting the virus. CHIKV is spread by two mosquito vectors, Aedes albopictus and Aedes aegypti, with increased presence around the globe. In this study, we report global spread of a CHIKV strain that carries two mutations that have been suggested to increase this virus ability to infect the Aedes aegypti mosquito, as well as to increase CHIKVs ability to be transmitted by this vector. We show that this strain appeared sometime between 2005 and 2008, most probably in India, and has now spread to Africa, Asia, and Australia. We show that this strain is capable of driving large outbreaks of CHIKV in the human population, causing recent major outbreaks in Kenya, Pakistan, India and Bangladesh. Thus, our results stress the importance of monitoring this strains global spread, as well as the need of improved vector control strategies in the areas of Aedes aegypti prevalence.

evolutionary biology↗

bioRxiv2017

The high burden of dengue and chikungunya in southern coastal Ecuador: Epidemiology, clinical presentation, and phylogenetics from a prospective study in Machala in 2014 and 2015

Here we report the findings from the first two years of an arbovirus surveillance study conducted in Machala, Ecuador, a dengue endemic region (2014-2015). Patients with suspected dengue virus (DENV) infections (index cases, n=324) were referred from five Ministry of Health clinical sites. A subset of DENV positive index cases (n = 44) were selected, and individuals from the index household and four neighboring homes within 200-meters were recruited (n = 400). Individuals who entered the study, other than index cases, are referred to as associates. In 2014, 70.9% of index cases and 35.6% of associates had acute or recent DENV infections. In 2015, 28.3% of index cases and 12.8% of associates had acute or recent DENV infections. For every DENV infection captured by passive surveillance, we detected an additional three acute or recent DENV infections in associates. Of associates with acute DENV infections, 68% reported dengue-like symptoms, with the highest prevalence of symptomatic acute infections in children under 10 years of age. The first chikungunya virus (CHIKV) infections were detected on epidemiological week 12 in 2015. 43.1% of index cases and 3.5% of associates had acute CHIKV infections. No Zika virus infections were detected. Phylogenetic analyses of isolates of DENV from 2014 revealed genetic relatedness and shared ancestry of DENV1, DENV2 and DENV4 genomes from Ecuador with those from Venezuela and Colombia, indicating presence of viral flow between Ecuador and surrounding countries. Enhanced surveillance studies, such as this, provide high-resolution data on symptomatic and inapparent infections across the population.

epidemiology↗