Virus Discovery and Characterization using Next-Generation Sequencing

__Abstract__ Infectious diseases can be caused by a wide variety of pathogens, including viruses, bacteria, protozoa, fungi, helminths and prions. For numerous known pathogens, the disease incidence has increased over the last few decades. In addition, several previously unknown pathogens have recently been discovered, adding up to the impact of infectious diseases in humans and animals. The term “emerging infectious diseases” has been coined to describe infectious diseases caused by newly discovered pathogens and by known pathogens that have recently become more widespread. Much emerging infectious disease research focuses on the importance of the interplay between humans, animals, and the environment in relation to health and disease. Because of industrialization, globalization, urbanization, deforestation, extensive commercialization of agriculture, and various other factors, the incidence of exposure of both animal and human populations to pathogens has increased. This results in increased risks of a wide range of zoonotic diseases, i.e. diseases that can be passed from animals to humans. Animals are known to represent the origin of up to 75% of the emerging infectious diseases in humans. Examples of such zoonoses are the human immunodeficiency virus (HIV) and Ebola virus that were transmitted from non-human primates, and Lyme disease that is transmitted from animals by ticks. On many occasions, humans are infected through an intermediate animal host rather than the original “reservoir” species. Hendra virus, Nipah virus, and SARS coronavirus (SARS-CoV) are pathogens that originate from bats, but infected humans through horses, pigs, and palm civets, respectively. The rate of contact between a pathogen reservoir and a novel host species is an important factor for successful interspecies transmission. Unfortunately, due to the changes described above, these contact rates have increase. From 1980 onwards, the number of infectious outbreaks in both humans and animals have increased. Nearly 40 infectious diseases in humans have been identified that were unknown a few decades ago. In addition, many known infectious diseases that were thought to be under control have recently re-emerged. According to the WHO, “ It would be extremely naïve and complacent to assume that there will be no other disease like AIDS, Ebola, or SARS, sooner or later”. In response to these words, several international initiatives were launched to counteract this threat, including a European consortium that leads the framework 7 program “EMPERIE”. Many of the recent outbreaks of infectious disease in humans and animals were caused by RNA-viruses, as summarized in figure 1. RNA viruses are among the most variable and taxonomically diverse pathogens, with a wide variety of host species

Characterization of the Mouse Neuroinvasiveness of Selected European Strains of West Nile Virus

West Nile virus (WNV) has caused outbreaks and sporadic infections in Central, Eastern and Mediterranean Europe for over 45 years. Most strains responsible for the European and Mediterranean basin outbreaks are classified as lineage 1. In recent years, WNV strains belonging to lineage 1 and 2 have been causing outbreaks of neuroinvasive disease in humans in countries such as Italy, Hungary and Greece, while mass mortality among birds was not reported. This study characterizes three European strains of WNV isolated in Italy (FIN and Ita09) and Hungary (578/10) in terms of in vitro replication kinetics on neuroblastoma cells, LD50 values in C57BL/6 mice, median day mortality, cumulative mortality, concentration of virus in the brain and spinal cord, and the response to infection in the brain. Overall, the results indicate that strains circulating in Europe belonging to both lineage 1 and 2 are highly virulent and that Ita09 and 578/10 are more neurovirulent compared to the FIN strain

Schatten van grasopbrengst op basis van spectrale reflectie, grashoogte en modellering : Onderzoeksresultaten van een maaiproef op zand- klei en veengrond 2016-2017

For dairy farmers a good estimate of the current grass yield is essential for grassland planning. To see to what extent grass yield and nitrogen content can be measured with remote sensing, spectral reflection measurements were compared in a field trial with dry matter yields and nitrogen content of mown grass (‘ground truth’ data). These yields and contents were also used to validate a model-based estimate of the grass yield and the nitrogen content or crude protein content. A practical comparison has also been made with grass height measurements. Reflection measurements gave a rough estimate of the dry matter yield. By making a distinction between location and by including basic growth data, the estimate improved considerably. A model-based growth prediction gave a better estimate of the dry matter yield and improved even further in combination with reflection measurements or grass height. With the current techniques, nitrogen content of grass appears to be difficult to measure and predict

Quasispecies composition and evolution of a typical Zika virus clinical isolate from Suriname

Molecular Technology and Informatics for Personalised Medicine and Healt

Functional properties of measles virus proteins derived from a subacute sclerosing panencephalitis patient who received repeated remdesivir treatments

Subacute sclerosing panencephalitis (SSPE) is a rare but fatal late neurological complication of measles, caused by persistent measles virus (MeV) infection of the central nervous system. There are no drugs approved for the treatment of SSPE. Here, we followed the clinical progression of a 5-year-old SSPE patient after treatment with the nucleoside analog remdesivir, conducted a post-mortem evaluation of the patient’s brain, and characterized the MeV detected in the brain. The quality of life of the patient transiently improved after the first two courses of remdesivir, but a third course had no further clinical effect, and the patient eventually succumbed to his condition. Post-mortem evaluation of the brain displayed histopathological changes including loss of neurons and demyelination paired with abundant presence of MeV RNA-positive cells throughout the brain. Next-generation sequencing of RNA isolated from the brain revealed a complete MeV genome with mutations that are typically detected in SSPE, characterized by a hypermutated M gene. Additional mutations were detected in the polymerase (L) gene, which were not associated with resistance to remdesivir. Functional characterization showed that mutations in the F gene led to a hyperfusogenic phenotype predominantly mediated by N465I. Additionally, recombinant wild-type-based MeV with the SSPE-F gene or the F gene with the N465I mutation was no longer lymphotropic but instead efficiently disseminated in neural cultures. Altogether, this case encourages further investigation of remdesivir as a potential treatment of SSPE and highlights the necessity to functionally understand SSPE-causing MeV.</p

Helpers compensate for age‐related declines in parental care and offspring survival in a cooperatively breeding bird

Offspring from elderly parents often have lower survival due to parental senescence. In cooperatively breeding species, where offspring care is shared between breeders and helpers, the alloparental care provided by helpers is predicted to mitigate the impact of parental senescence on offspring provisioning and, subsequently, offspring survival. We test this prediction using data from a long‐term study on cooperatively breeding Seychelles warblers (Acrocephalus sechellensis). We find that the nestling provisioning rate of female breeders declines with their age. Further, the total brood provisioning rate and the first‐year survival probability of offspring decline progressively with age of the female breeder, but these declines are mitigated when helpers are present. This effect does not arise because individual helpers provide more care in response to the lower provisioning of older dominant females, but because older female breeders have recruited more helpers, thereby receiving more overall care for their brood. We do not find such effects for male breeders. These results indicate that alloparental care can alleviate the fitness costs of senescence for breeders, which suggests an interplay between age and cooperative breeding

Functional properties of measles virus proteins derived from a subacute sclerosing panencephalitis patient who received repeated remdesivir treatments

Subacute sclerosing panencephalitis (SSPE) is a rare but fatal late neurological complication of measles, caused by persistent measles virus (MeV) infection of the central nervous system. There are no drugs approved for the treatment of SSPE. Here, we followed the clinical progression of a 5-year-old SSPE patient after treatment with the nucleoside analog remdesivir, conducted a post-mortem evaluation of the patient’s brain, and characterized the MeV detected in the brain. The quality of life of the patient transiently improved after the first two courses of remdesivir, but a third course had no further clinical effect, and the patient eventually succumbed to his condition. Post-mortem evaluation of the brain displayed histopathological changes including loss of neurons and demyelination paired with abundant presence of MeV RNA-positive cells throughout the brain. Next-generation sequencing of RNA isolated from the brain revealed a complete MeV genome with mutations that are typically detected in SSPE, characterized by a hypermutated M gene. Additional mutations were detected in the polymerase (L) gene, which were not associated with resistance to remdesivir. Functional characterization showed that mutations in the F gene led to a hyperfusogenic phenotype predominantly mediated by N465I. Additionally, recombinant wild-type-based MeV with the SSPE-F gene or the F gene with the N465I mutation was no longer lymphotropic but instead efficiently disseminated in neural cultures. Altogether, this case encourages further investigation of remdesivir as a potential treatment of SSPE and highlights the necessity to functionally understand SSPE-causing MeV.</p

Performance of five metagenomic classifiers for virus pathogen detection using respiratory samples from a clinical cohort

Viral metagenomics is increasingly applied in clinical diagnostic settings for detection of pathogenic viruses. While several benchmarking studies have been published on the use of metagenomic classifiers for abundance and diversity profiling of bacterial populations, studies on the comparative performance of the classifiers for virus pathogen detection are scarce. In this study, metagenomic data sets (n = 88) from a clinical cohort of patients with respiratory complaints were used for comparison of the performance of five taxonomic classifiers: Centrifuge, Clark, Kaiju, Kraken2, and Genome Detective. A total of 1144 positive and negative PCR results for a total of 13 respiratory viruses were used as gold standard. Sensitivity and specificity of these classifiers ranged from 83 to 100% and 90 to 99%, respectively, and was dependent on the classification level and data pre-processing. Exclusion of human reads generally resulted in increased specificity. Normalization of read counts for genome length resulted in a minor effect on overall performance, however it negatively affected the detection of targets with read counts around detection level. Correlation of sequence read counts with PCR Ct-values varied per classifier, data pre-processing (R-2 range 15.1-63.4%), and per virus, with outliers up to 3 log(10) reads magnitude beyond the predicted read count for viruses with high sequence diversity. In this benchmarking study, sensitivity and specificity were within the ranges of use for diagnostic practice when the cut-off for defining a positive result was considered per classifier.Molecular Epidemiolog

Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction

We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.5-6.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation

A Family-Wide RT-PCR Assay for Detection of Paramyxoviruses and Application to a Large-Scale Surveillance Study

Family-wide molecular diagnostic assays are valuable tools for initial identification of viruses during outbreaks and to limit costs of surveillance studies. Recent discoveries of paramyxoviruses have called for such assay that is able to detect all known and unknown paramyxoviruses in one round of PCR amplification. We have developed a RT-PCR assay consisting of a single degenerate primer set, able to detect all members of the Paramyxoviridae family including all virus genera within the subfamilies Paramyxovirinae and Pneumovirinae. Primers anneal to domain III of the polymerase gene, with the 3′ end of the reverse primer annealing to the conserved motif GDNQ, which is proposed to be the active site for nucleotide polymerization. The assay was fully optimized and was shown to indeed detect all available paramyxoviruses tested. Clinical specimens from hospitalized patients that tested positive for known paramyxoviruses in conventional assays were also detected with the novel family-wide test. A high-throughput fluorescence-based RT-PCR version of the assay was developed for screening large numbers of specimens. A large number of samples collected from wild birds was tested, resulting in the detection of avian paramyxoviruses type 1 in both barnacle and white-fronted geese, and type 8 in barnacle geese. Avian metapneumovirus type C was found for the first time in Europe in mallards, greylag geese and common gulls. The single round family-wide RT-PCR assay described here is a useful tool for the detection of known and unknown paramyxoviruses, and screening of large sample collections from humans and animals