A Novel Approach to Sequencing West Nile Virus Genome using IDT xGen and Illumina MiniSeq

The West Nile virus (WNV) has been identified as a cause of mosquito-borne illness in the continental United States (CDC, 2023). It is a member of the Japanese encephalitis antigenic complex of the Flaviviridae family. WNV is predominantly maintained in an enzootic transmission cycle between Culex species mosquitoes and birds as vertebrate hosts. Humans and horses are considered WNV incidental hosts (a.k.a dead-end hosts) and cannot be transferred to another host. Within the last five years (2018-2022), 8,386 WNV human infection cases have been recorded. In addition, 90 WNV human infection cases have been reported in 17 different states this year. Nebraska has recorded the 4th highest total number of cases since WNV was introduced into the US. Monitoring the genetic variability of WNV will allow researchers to elucidate transmission patterns and ultimately incorporate WNV genomics into estimates of human risk. Understanding virus evolution through time requires an in-depth understanding of genomics. This research project aims to develop a more efficient and effective method for sequencing WNV genomes to better understand evolution. In addition, a novel approach was adopted to sequence WNV from mosquito pools collected in Nebraska. The results of this research suggested that the IDT xGen could potentially be used to sequence WNV from mosquito pools.https://digitalcommons.unmc.edu/surp2023/1010/thumbnail.jp

De novo Assembly of the Brugia malayi Genome Using Long Reads from a Single MinION Flowcell

Filarial nematode infections cause a substantial global disease burden. Genomic studies of filarial worms can improve our understanding of their biology and epidemiology. However, genomic information from field isolates is limited and available reference genomes are often discontinuous. Single molecule sequencing technologies can reduce the cost of genome sequencing and long reads produced from these devices can improve the contiguity and completeness of genome assemblies. In addition, these new technologies can make generation and analysis of large numbers of field isolates feasible. In this study, we assessed the performance of the Oxford Nanopore Technologies MinION for sequencing and assembling the genome of Brugia malayi, a human parasite widely used in filariasis research. Using data from a single MinION flowcell, a 90.3 Mb nuclear genome was assembled into 202 contigs with an N50 of 2.4 Mb. This assembly covered 96.9% of the well-defined B. malayi reference genome with 99.2% identity. The complete mitochondrial genome was obtained with individual reads and the nearly complete genome of the endosymbiotic bacteria Wolbachia was assembled alongside the nuclear genome. Long-read data from the MinION produced an assembly that approached the quality of a well-established reference genome using comparably fewer resources

Xenosurveillance reflects traditional sampling techniques for the identification of human pathogens: A comparative study in West Africa

BACKGROUND: Novel surveillance strategies are needed to detect the rapid and continuous emergence of infectious disease agents. Ideally, new sampling strategies should be simple to implement, technologically uncomplicated, and applicable to areas where emergence events are known to occur. To this end, xenosurveillance is a technique that makes use of blood collected by hematophagous arthropods to monitor and identify vertebrate pathogens. Mosquitoes are largely ubiquitous animals that often exist in sizable populations. As well, many domestic or peridomestic species of mosquitoes will preferentially take blood-meals from humans, making them a unique and largely untapped reservoir to collect human blood. METHODOLOGY/PRINCIPAL FINDINGS: We sought to take advantage of this phenomenon by systematically collecting blood-fed mosquitoes during a field trail in Northern Liberia to determine whether pathogen sequences from blood engorged mosquitoes accurately mirror those obtained directly from humans. Specifically, blood was collected from humans via finger-stick and by aspirating bloodfed mosquitoes from the inside of houses. Shotgun metagenomic sequencing of RNA and DNA derived from these specimens was performed to detect pathogen sequences. Samples obtained from xenosurveillance and from finger-stick blood collection produced a similar number and quality of reads aligning to two human viruses, GB virus C and hepatitis B virus. CONCLUSIONS/SIGNIFICANCE: This study represents the first systematic comparison between xenosurveillance and more traditional sampling methodologies, while also demonstrating the viability of xenosurveillance as a tool to sample human blood for circulating pathogens

Determining Schistosoma haematobium Population Structures in Ethiopia

Schistosomiasis, also known as bilharzia, is a Neglected Tropical Disease caused by parasitic helminths that affects over 240 million people around the world. Praziquantel has been used to treat individuals infected with schistosomiasis through Mass Drug Administration (MDA) programs but a recent reduction in its efficacy has been observed, creating concern that the parasite will evolve to become resistant to the chemotherapy drug. Monitoring changes in the population structure of Schistosoma haematobium using microsatellite markers can be a useful metric to determine praziquantel efficacy since variations in the repeat sequences of microsatellites indicate genetic diversity. Since little is known about the population structure of S. haematobium–despite urogenital schistosomiasis being a pressing issue in Ethiopia–18 microsatellite multiplex assays developed in a prior study were tested on stock DNA to validate them for use to study the effects of praziquantel on parasitic S. haematobium in Ethiopia. Through a combination of bioinformatic analysis, PCR, and Next Generation Sequencing on the MinION, 13 of the 18 microsatellite markers were validated, highlighting the importance of developing microsatellite multiplex assays not only based on length distribution, but also based on Next Generation Sequencing data.https://digitalcommons.unmc.edu/surp2023/1015/thumbnail.jp

Maporal Hantavirus Causes Mild Pathology in Deer Mice (\u3ci\u3ePeromyscus maniculatus\u3c/i\u3e)

Rodent-borne hantaviruses can cause two human diseases with many pathological similarities: hantavirus cardiopulmonary syndrome (HCPS) in the western hemisphere and hemorrhagic fever with renal syndrome in the eastern hemisphere. Each virus is hosted by specific reservoir species without conspicuous disease. HCPS-causing hantaviruses require animal biosafety level-4 (ABSL-4) containment, which substantially limits experimental research of interactions between the viruses and their reservoir hosts. Maporal virus (MAPV) is a South American hantavirus not known to cause disease in humans, thus it can be manipulated under ABSL-3 conditions. The aim of this study was to develop an ABSL-3 hantavirus infection model using the deer mouse (Peromyscus maniculatus), the natural reservoir host of Sin Nombre virus (SNV), and a virus that is pathogenic in another animal model to examine immune response of a reservoir host species. Deer mice were inoculated with MAPV, and viral RNA was detected in several organs of all deer mice during the 56 day experiment. Infected animals generated both nucleocapsid-specific and neutralizing antibodies. Histopathological lesions were minimal to mild with the peak of the lesions detected at 7–14 days postinfection, mainly in the lungs, heart, and liver. Low to modest levels of cytokine gene expression were detected in spleens and lungs of infected deer mice, and deer mouse primary pulmonary cells generated with endothelial cell growth factors were susceptible to MAPV with viral RNA accumulating in the cellular fraction compared to infected Vero cells. Most features resembled that of SNV infection of deer mice, suggesting this model may be an ABSL-3 surrogate for studying the host response of a New World hantavirus reservoir

Maporal Hantavirus Causes Mild Pathology in Deer Mice (\u3ci\u3ePeromyscus maniculatus\u3c/i\u3e)

Rodent-borne hantaviruses can cause two human diseases with many pathological similarities: hantavirus cardiopulmonary syndrome (HCPS) in the western hemisphere and hemorrhagic fever with renal syndrome in the eastern hemisphere. Each virus is hosted by specific reservoir species without conspicuous disease. HCPS-causing hantaviruses require animal biosafety level-4 (ABSL-4) containment, which substantially limits experimental research of interactions between the viruses and their reservoir hosts. Maporal virus (MAPV) is a South American hantavirus not known to cause disease in humans, thus it can be manipulated under ABSL-3 conditions. The aim of this study was to develop an ABSL-3 hantavirus infection model using the deer mouse (Peromyscus maniculatus), the natural reservoir host of Sin Nombre virus (SNV), and a virus that is pathogenic in another animal model to examine immune response of a reservoir host species. Deer mice were inoculated with MAPV, and viral RNA was detected in several organs of all deer mice during the 56 day experiment. Infected animals generated both nucleocapsid-specific and neutralizing antibodies. Histopathological lesions were minimal to mild with the peak of the lesions detected at 7–14 days postinfection, mainly in the lungs, heart, and liver. Low to modest levels of cytokine gene expression were detected in spleens and lungs of infected deer mice, and deer mouse primary pulmonary cells generated with endothelial cell growth factors were susceptible to MAPV with viral RNA accumulating in the cellular fraction compared to infected Vero cells. Most features resembled that of SNV infection of deer mice, suggesting this model may be an ABSL-3 surrogate for studying the host response of a New World hantavirus reservoir

Sequencing SARS-CoV-2 Genomes From Saliva

Genomic sequencing is crucial to understanding the epidemiology and evolution of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Often, genomic studies rely on remnant diagnostic material, typically nasopharyngeal (NP) swabs, as input into whole-genome SARS-CoV-2 next-generation sequencing pipelines. Saliva has proven to be a safe and stable specimen for the detection of SARS-CoV-2 RNA via traditional diagnostic assays; however, saliva is not commonly used for SARS-CoV-2 sequencing. Using the ARTIC Network amplicon-generation approach with sequencing on the Oxford Nanopore MinION, we demonstrate that sequencing SARS-CoV-2 from saliva produces genomes comparable to those from NP swabs, and that RNA extraction is necessary to generate complete genomes from saliva. In this study, we show that saliva is a useful specimen type for genomic studies of SARS-CoV-2

Investigation of a SARS-CoV-2 B.1.1.529 (Omicron) Variant Cluster - Nebraska, November-December 2021

The B.1.1.529 (Omicron) variant of SARS-CoV-2 (the virus that causes COVID-19) was first detected in specimens collected on November 11, 2021, in Botswana and on November 14 in South Africa;* the first confirmed case of Omicron in the United States was identified in California on December 1, 2021 (1). On November 29, the Nebraska Department of Health and Human Services was notified of six probable cases† of COVID-19 in one household, including one case in a man aged 48 years (the index patient) who had recently returned from Nigeria. Given the patient\u27s travel history, Omicron infection was suspected. Specimens from all six persons in the household tested positive for SARS-CoV-2 by reverse transcription-polymerase chain reaction (RT-PCR) testing on December 1, and the following day genomic sequencing by the Nebraska Public Health Laboratory identified an identical Omicron genotype from each specimen (Figure). Phylogenetic analysis was conducted to determine if this cluster represented an independent introduction of Omicron into the United States, and a detailed epidemiologic investigation was conducted. This activity was reviewed by CDC and was conducted consistent with applicable federal law and CDC policy.§

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