Deep sequencing of norovirus genomes defines evolutionary patterns in an urban tropical setting.

UNLABELLED: Norovirus is a highly transmissible infectious agent that causes epidemic gastroenteritis in susceptible children and adults. Norovirus infections can be severe and can be initiated from an exceptionally small number of viral particles. Detailed genome sequence data are useful for tracking norovirus transmission and evolution. To address this need, we have developed a whole-genome deep-sequencing method that generates entire genome sequences from small amounts of clinical specimens. This novel approach employs an algorithm for reverse transcription and PCR amplification primer design using all of the publically available norovirus sequence data. Deep sequencing and de novo assembly were used to generate norovirus genomes from a large set of diarrheal patients attending three hospitals in Ho Chi Minh City, Vietnam, over a 2.5-year period. Positive-selection analysis and direct examination of protein changes in the virus over time identified codons in the regions encoding proteins VP1, p48 (NS1-2), and p22 (NS4) under positive selection and expands the known targets of norovirus evolutionary pressure. IMPORTANCE: The high transmissibility and rapid evolutionary rate of norovirus, combined with a short-lived host immune responses, are thought to be the reasons why the virus causes the majority of pediatric viral diarrhea cases. The evolutionary patterns of this RNA virus have been described in detail for only a portion of the virus genome and never for a virus from a detailed urban tropical setting. We provide a detailed sequence description of the noroviruses circulating in three Ho Chi Minh City hospitals over a 2.5-year period. This study identified patterns of virus change in known sites of host immune response and identified three additional regions of the virus genome under selection that were not previously recognized. In addition, the method described here provides a robust full-genome sequencing platform for community-based virus surveillance

Measles whole-genome sequencing

This is an optimised protocol for PCR amplification of the Measles virus (MV) genome using specific primers designed for B3 genotype. The method generates cDNA amplicons suitable for whole genome sequencing using the MinION platform. The method covers the entire ca. 16,000 nt MV genome in 8 overlapping amplicons of about 2200 bp (see Figure below). Both the reverse transcription and PCR steps are performed in two separate reactions to avoid short overlapping PCR products. The method has worked successfully with MV directly from oral fluid samples and with tissue culture passaged material. Primer sequences can be found at the following GitHub site: https://github.com/mlcotten13/Measles_primer

Unique protein features of SARS-CoV-2 relative to other Sarbecoviruses

Defining the unique properties of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein sequences has potential to explain the range of Coronavirus Disease 2019 severity. To achieve this we compared proteins encoded by all Sarbecoviruses using profile Hidden Markov Model similarities to identify protein features unique to SARS-CoV-2. Consistent with previous reports, a small set of bat- and pangolin-derived Sarbecoviruses show the greatest similarity to SARS-CoV-2 but are unlikely to be the direct source of SARS-CoV-2. Three proteins (nsp3, spike, and orf9) showed regions differing between the bat Sarbecoviruses and SARS-CoV-2 and indicate virus protein features that might have evolved to support human infection and/or transmission. Spike analysis identified all regions of the protein that have tolerated change and revealed that the current SARS-CoV-2 variants of concern have sampled only a fraction (∼31 per cent) of the possible spike domain changes which have occurred historically in Sarbecovirus evolution. This result emphasises the evolvability of these coronaviruses and the potential for further change in virus replication and transmission properties over the coming years

Characterization of Norovirus and Other Human Enteric Viruses in Sewage and Stool Samples Through Next-Generation Sequencing.

This study aimed to optimize a method to identify human enteric viruses in sewage and stool samples using random primed next-generation sequencing. We tested three methods, two employed virus enrichment based on the binding properties of the viral capsid using pig-mucin capture or by selecting viral RNA prior to library preparation through a capture using the SureSelect target enrichment. The third method was based on a non-specific biophysical precipitation with polyethylene glycol. Full genomes of a number of common human enteric viruses including norovirus, rotavirus, husavirus, enterovirus and astrovirus were obtained. In stool samples full norovirus genome were detected as well as partial enterovirus genome. A variety of norovirus sequences was detected in sewage samples, with genogroup II being more prevalent. Interestingly, the pig-mucin capture enhanced not only the recovery of norovirus and rotavirus but also recovery of astrovirus, sapovirus and husavirus. Documenting sewage virome using these methods provides information for molecular epidemiology and may be useful in developing strategies to prevent further spread of viruses

Increased resolution of African swine fever virus genome patterns based on profile HMMs of protein domains.

African swine fever virus (ASFV), belonging to the Asfarviridae family, was originally described in Africa almost 100?years ago and is now spreading uncontrolled across Europe and Asia and threatening to destroy the domestic pork industry. Neither effective antiviral drugs nor protective vaccines are currently available. Efforts to understand the basis for viral pathogenicity and the development of attenuated potential vaccine strains are complicated by the large and complex nature of the ASFV genome. We report here a novel alignment-free method of documenting viral diversity based on profile hidden Markov model domains on a genome scale. The method can be used to infer genomic relationships independent of genome alignments and also reveal ASFV genome sequence differences that determine the presence and characteristics of functional protein domains in the virus. We show that the method can quickly identify differences and shared patterns between virulent and attenuated ASFV strains and will be a useful tool for developing much-needed vaccines and antiviral agents to help control this virus. The tool is rapid to run and easy to implement, readily available as a simple Docker image

Complete Genome Characterization of Eight Human Parainfluenza Viruses from the Netherlands.

We report the complete genome sequences of eight human parainfluenza viruses (HPIV) belonging to Human respirovirus 1 (HPIV-1), Human respirovirus 3 (HPIV-3), Human rubulavirus 2 (HPIV-2), and Human rubulavirus 4 (HPIV-4). The genome sequences were generated using random-primed next-generation sequencing and represent the first HPIV full-genome sequences from the Netherlands

Neutrophils in cancer: neutral no more

Neutrophils are indispensable antagonists of microbial infection and facilitators of wound healing. In the cancer setting, a newfound appreciation for neutrophils has come into view. The traditionally held belief that neutrophils are inert bystanders is being challenged by the recent literature. Emerging evidence indicates that tumours manipulate neutrophils, sometimes early in their differentiation process, to create diverse phenotypic and functional polarization states able to alter tumour behaviour. In this Review, we discuss the involvement of neutrophils in cancer initiation and progression, and their potential as clinical biomarkers and therapeutic targets

Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia.

BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients

High rate nitrogen removal by ANAMMOX internal circulation reactor (IC) for old landfill leachate treatment

© 2017 Elsevier Ltd This study aimed to evaluate the performance of a high rate nitrogen removal lab-scale ANAMMOX reactor, namely Internal Circulation (IC) reactor, for old landfill leachate treatment. The reactor was operated with pre-treated leachate from a pilot Partial Nitritation Reactor (PNR) using a high nitrogen loading rate ranging from 2 to 10 kg N m−3 d−1. High rate removal of nitrogen (9.52 ± 1.11 kg N m−3 d−1) was observed at an influent nitrogen concentration of 1500 mg N L−1. The specific ANAMMOX activity was found to be 0.598 ± 0.026 gN2-N gVSS−1 d−1. Analysis of ANAMMOX granules suggested that 0.5–1.0 mm size granular sludge was the dominant group. The results of DNA analysis revealed that Candidatus Kueneniastuttgartiensis was the dominant species (37.45%) in the IC reactor, whereas other species like uncultured Bacteroidetes bacterium only constituted 5.37% in the system, but they were still responsible for removing recalcitrant organic matter