Coronavirus (CoV) infection of humans is usually not associated with severe disease. However, discovery of the severe acute respiratory syndrome (SARS) CoV revealed that highly pathogenic human CoVs (HCoVs) can evolve. The identification and characterization of new HCoVs is, therefore, an important task. Recently, a HCoV termed NL63 was discovered in patients with respiratory tract illness. Here, cell tropism and receptor usage of HCoV-NL63 were analyzed. The NL63 spike (S) protein mediated infection of different target cells compared with the closely related 229E-S protein but facilitated entry into cells known to be permissive to SARS-CoV-S-driven infection. An analysis of receptor engagement revealed that NL63-S binds angiotensin-converting enzyme (ACE) 2, the receptor for SARS-CoV, and HCoV-NL63 uses ACE2 as a receptor for infection of target cells. Potent neutralizing activity directed against NL63- but not 229E-S protein was detected in virtually all sera from patients 8 years of age or older, suggesting that HCoV-NL63 infection of humans is common and usually acquired during childhood. Here, we show that SARS-CoV shares its receptor ACE2 with HCoV-NL63. Because the two viruses differ dramatically in their ability to induce disease, analysis of HCoV-NL63 might unravel pathogenicity factors in SARS-CoV. The frequent HCoV-NL63 infection of humans suggests that highly pathogenic variants have ample opportunity to evolve, underlining the need for vaccines against HCoVs.
Anelloviruses (AVs) are found in the vast majority of the human population and are most probably part of a healthy virome. These viruses infect humans in the early stage of life, however, the characteristics of the first colonizing AVs are still unknown. We screened a collection of 107 blood samples from children between 0.4 and 64.8 months of age for the presence of three AV genera: the Alpha-, Beta- and Gammatorquevirus. The youngest child that was positive for AV was 1.2 months old, and a peak in prevalence (100% of samples positive) was reached between the twelfth and eighteenth months of life. Intriguingly, the beta- and gammatorqueviruses were detected most at the early stage of life (up to 12 months), whereas alphatorqueviruses, the most common AVs in adults, increased in prevalence in children older than 12 months. To determine whether that order of colonization may be related to oral transmission and unequal presence of AV genera in breast milk, we examined 63 breast milk samples. Thirty-two percent of the breast milk samples were positive in a qPCR detecting beta- and gammatorqueviruses, while alphatorqueviruses were detected in 10% of the samples, and this difference was significant (p = 0.00654). In conclusion, we show that beta- and gammatorqueviruses colonize humans in the first months of life and that breastfeeding could play a role in AV transmission.
VIDISCA is a next-generation sequencing (NGS) library preparation method designed to enrich viral nucleic acids from samples before highly-multiplexed low depth sequencing. Reliable detection of known viruses and discovery of novel divergent viruses from NGS data require dedicated analysis tools that are both sensitive and accurate. Existing software was utilised to design a new bioinformatic workflow for high-throughput detection and discovery of viruses from VIDISCA data. The workflow leverages the VIDISCA library preparation molecular biology, specifically the use of Mse1 restriction enzyme which produces biological replicate library inserts from identical genomes. The workflow performs total metagenomic analysis for classification of non-viral sequence including parasites and host, and separately carries out virus specific analyses. Ribosomal RNA sequence is removed to increase downstream analysis speed and remaining reads are clustered at 100% identity. Known and novel viruses are sensitively detected via alignment to a virus-only protein database, and false positives are removed. A new cluster-profiling analysis takes advantage of the viral biological replicates produced by Mse1 digestion, using read clustering to flag the presence of short genomes at very high copy number. Importantly, this analysis ensures that highly repeated sequences are identified even if no homology is detected, as is shown here with the detection of a novel gokushovirus genome from human faecal matter. The workflow was validated using read data derived from serum and faeces samples taken from HIV-1 positive adults, and serum samples from pigs that were infected with atypical porcine pestivirus.
A simple method for the isolation and subsequent detection of human immunodeficiency virus type 1 (HIV-1) RNA from feces is described. Viral RNA was isolated by the method developed by Boom et al. (R. Boom, C.J.A. Sol, M.M.M. Salimans, C.L. Jansen, P.M.E. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990), which was adapted for feces. HIV-1 RNA was detected by reverse transcription (RT) followed by a nested PCR encompassing the V3 region. Reconstruction experiments revealed that the efficiencies of the extraction technique and the subsequent RT-PCR were not considerably affected by the varied composition of feces. The method was applied on fecal specimens from 18 HIV-1-infected individuals, among which were samples that had been stored for 9 years. It appeared that HIV-1 RNA was detectable in the feces of 12 persons (67%). Viral RNA was present in the feces of persons who fulfilled the criteria for CDC class II and CDC class III HIV infection as well as in patients who were diagnosed with AIDS (CDC class IV). Direct sequencing of amplimers obtained from paired fecal and serum specimens showed that differences in sequence heterogeneity existed. In one patient a remarkable difference in the HIV-1 sequences between isolates from feces and serum was observed. In conclusion, HIV-1 RNA is frequently present in the feces of HIV-1-infected individuals, and in some cases the HIV-1 subpopulation in feces differs from the HIV-1 subpopulation in serum.
Abstract Background Gastrointestinal symptoms, in particular diarrhoea, are common in non-treated HIV-1 infected individuals. Although various enteric pathogens have been implicated, the aetiology of diarrhoea remains unexplained in a large proportion of HIV-1 infected patients. Our aim is to identify the cause of diarrhoea for patients that remain negative in routine diagnostics. Methods In this study stool samples of 196 HIV-1 infected persons, including 29 persons with diarrhoea, were examined for enteropathogens and HIV-1. A search for unknown and unexpected viruses was performed using virus discovery cDNA-AFLP combined with Roche-454 sequencing (VIDISCA-454). Results HIV-1 RNA was detected in stool of 19 patients with diarrhoea (66%) compared to 75 patients (45%) without diarrhoea. In 19 of the 29 diarrhoea cases a known enteropathogen could be identified (66%). Next to these known causative agents, a range of recently identified viruses was identified via VIDISCA-454: cosavirus, Aichi virus, human gyrovirus, and non-A non-B hepatitis virus. Moreover, a novel virus was detected which was named immunodeficiency-associated stool virus (IASvirus). However, PCR based screening for these viruses showed that none of these novel viruses was associated with diarrhoea. Notably, among the 34% enteropathogen-negative cases, HIV-1 RNA shedding in stool was more frequently observed (80%) compared to enteropathogen-positive cases (47%), indicating that HIV-1 itself is the most likely candidate to be involved in diarrhoea. Conclusion Unexplained diarrhoea in HIV-1 infected patients is probably not caused by recently described or previously unknown pathogens, but it is more likely that HIV-1 itself plays a role in intestinal mucosal abnormalities which leads to diarrhoea.
Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.
We have developed a full genome virus detection process that combines sensitive nucleic acid preparation optimised for virus identification in fecal material with Illumina MiSeq sequencing and a novel post-sequencing virus identification algorithm. Enriched viral nucleic acid was converted to double-stranded DNA and subjected to Illumina MiSeq sequencing. The resulting short reads were processed with a novel iterative Python algorithm SLIM for the identification of sequences with homology to known viruses. De novo assembly was then used to generate full viral genomes. The sensitivity of this process was demonstrated with a set of fecal samples from HIV-1 infected patients. A quantitative assessment of the mammalian, plant, and bacterial virus content of this compartment was generated and the deep sequencing data were sufficient to assembly 12 complete viral genomes from 6 virus families. The method detected high levels of enteropathic viruses that are normally controlled in healthy adults, but may be involved in the pathogenesis of HIV-1 infection and will provide a powerful tool for virus detection and for analyzing changes in the fecal virome associated with HIV-1 progression and pathogenesis.
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