Influenza A H1N1/2009 virus that emerged from swine rapidly replaced the previous seasonal H1N1 virus. Although the early emergence and diversification of H1N1/2009 is well characterized, the ongoing evolutionary and global transmission dynamics of the virus remain poorly investigated. To address this we analyse >3,000 H1N1/2009 genomes, including 214 full genomes generated from our surveillance in Singapore, in conjunction with antigenic data. Here we show that natural selection acting on H1N1/2009 directly after introduction into humans was driven by adaptation to the new host. Since then, selection has been driven by immunological escape, with these changes corresponding to restricted antigenic diversity in the virus population. We also show that H1N1/2009 viruses have been subject to regular seasonal bottlenecks and a global reduction in antigenic and genetic diversity in 2014.

Influenza B viruses have circulated in humans for over 80 y, causing a significant disease burden. Two antigenically distinct lineages (“B/Victoria/2/87-like” and “B/Yamagata/16/88-like,” termed Victoria and Yamagata) emerged in the 1970s and have cocirculated since 2001. Since 2015 both lineages have shown unusually high levels of epidemic activity, the reasons for which are unclear. By analyzing over 12,000 influenza B virus genomes, we describe the processes enabling the long-term success and recent resurgence of epidemics due to influenza B virus. We show that following prolonged diversification, both lineages underwent selective sweeps across the genome and have subsequently taken alternate evolutionary trajectories to exhibit epidemic dominance, with no reassortment between lineages. Hemagglutinin deletion variants emerged concomitantly in multiple Victoria virus clades and persisted through epistatic mutations and interclade reassortment—a phenomenon previously only observed in the 1970s when Victoria and Yamagata lineages emerged. For Yamagata viruses, antigenic drift of neuraminidase was a major driver of epidemic activity, indicating that neuraminidase-based vaccines and cross-reactivity assays should be employed to monitor and develop robust protection against influenza B morbidity and mortality. Overall, we show that long-term diversification and infrequent selective sweeps, coupled with the reemergence of hemagglutinin deletion variants and antigenic drift of neuraminidase, are factors that contributed to successful circulation of diverse influenza B clades. Further divergence of hemagglutinin variants with poor cross-reactivity could potentially lead to circulation of 3 or more distinct influenza B viruses, further complicating influenza vaccine formulation and highlighting the urgent need for universal influenza vaccines.

Quantification of Epstein-Barr virus (EBV) cell-free DNA (cfDNA) is commonly used in clinical settings as a circulating biomarker in nasopharyngeal carcinoma (NPC), but there has been no comparison with circulating tumour cells (CTCs).Our study aims to compare the performance of CTC enumeration against EBV cfDNA quantitation through digital PCR (dPCR) and quantitative PCR. 74 plasma samples from 46 NPC patients at baseline and one month after radiotherapy with or without concurrent chemotherapy were analysed. CTCs were captured by microsieve technology and enumerated, while three different methods of EBV cfDNA quantification were applied, including an in-house qPCR assay for BamHI-W fragment, a CE-IVD qPCR assay (Sentosa ® ) and a dPCR (Clarity ™ ) assay for Epstein-Barr nuclear antigen 1 (EBNA1). EBV cfDNA quantitation by all workflows showed stronger correlation with clinical stage, radiological response and overall survival in comparison with CTC enumeration. The highest detection rate of EBV cfDNA in pre-treatment samples was seen with the BamHI-W qPCR assay (89%), followed by EBNA1-dPCR (85%) and EBNA1-qPCR (67%) assays. Overall, we show that EBV cfDNA outperforms CTC enumeration in correlation with clinical outcomes of NPC patients undergoing treatment. Techniques such as dPCR and target selection of BamHI-W may improve sensitivity for EBV cfDNA detection.Nasopharyngeal carcinoma (NPC) is a malignant cancer of the nasopharynx, which is particularly common in parts of Southern China, South East Asia and North Africa 1 . Due to high rates of Epstein-Barr virus (EBV) nucleic acid detection in NPC, non-invasive approaches to diagnosis have focused on EBV as a target [2][3][4] . Post-treatment Epstein-Barr virus (EBV) cell-free DNA (cfDNA) levels have been demonstrated to correlate with NPC prognosis and recurrence 5,6 . EBV cfDNA can be quantified in the form of EBV single-copy genes; EBNA1, LMP2 and Pol-1, or multiple-repeat fragments; BamHI-W 7 . As there are six to twenty copies of BamHI-W per EBV genome 8 , higher sensitivity is expected in BamHI-W quantification assays. However, the variability of BamHI-W copy numbers in different EBV isolates has been considered a challenges in assay comparison and standardization between laboratories 7, 8 . CTCs represent a circulating biomarker which has been extensively studied in many cancer types including breast, lung and colorectal cancer [9][10][11][12] . Due to challenges including platform costs and standardization, much

The ISI is a useful aid for decision-making during clinic consultation for patients presenting with symptoms suggestive of an RVI.

Norovirus (NoV) is the most common cause of sporadic and epidemic gastroenteritis, globally. This study aimed to investigate the molecular epidemiology of NoV-associated acute gastroenteritis in Singapore by classifying circulating NoV genotypes and genogroup II, genotype 4 (GII.4) variants between September 2004 and February 2011. The temporal dominance and antigenic variation within the circulating epidemic NoV GII.4 variants was also examined, in order to compare the trends in Singapore to those observed globally during the same period. A total of 312 of 1,060 fecal specimens were positive for NoV RNA, using a quantitative RT-PCR. In a subset (125 of 312) of NoV positive samples, the 5' end of ORF2 (region C) of the GI or GII NoV genome was amplified and sequenced. Subsequent phylogenetic analysis identified GII.4 was the most commonly identified genotype representing 80.8% (101/125) of NoV sequenced in this study. The predominant GII.4 variants in circulation during the 2004-2011 epidemic periods were Hunter 2004 (2004-2005), Den Haag 2006b (2006-2009), and New Orleans 2009 (2009-2011). Amino acid variation within the P2 domain of the major capsid protein, VP1, was followed longitudinally within the GII.4 lineage. A constant turnover of variant-specific amino acid change was observed, particularly within the antigenic epitopes A, C and E. In conclusion, this study has characterized the NoV strains in circulation in Singapore between 2004 and 2011. The molecular epidemiology and persistence of GII.4 pandemic NoV lineages in Singapore was similar to trends seen globally, with a noted absence of the Asia 2003 variant.