Characterisation of the immune repertoire of a humanised transgenic mouse through immunophenotyping and high-throughput sequencing.

Immunoglobulin loci-transgenic animals are widely used in antibody discovery and increasingly in vaccine response modelling. In this study, we phenotypically characterised B-cell populations from the Intelliselect Transgenic mouse (Kymouse) demonstrating full B-cell development competence. Comparison of the naïve B-cell receptor (BCR) repertoires of Kymice BCRs, naïve human, and murine BCR repertoires revealed key differences in germline gene usage and junctional diversification. These differences result in Kymice having CDRH3 length and diversity intermediate between mice and humans. To compare the structural space explored by CDRH3s in each species' repertoire, we used computational structure prediction to show that Kymouse naïve BCR repertoires are more human-like than mouse-like in their predicted distribution of CDRH3 shape. Our combined sequence and structural analysis indicates that the naïve Kymouse BCR repertoire is diverse with key similarities to human repertoires, while immunophenotyping confirms that selected naïve B cells are able to go through complete development.

Nosocomial transmission of influenza: A retrospective cross-sectional study using next generation sequencing at a hospital in England (2012-2014).

BACKGROUND: The extent of transmission of influenza in hospital settings is poorly understood. Next generation sequencing may improve this by providing information on the genetic relatedness of viral strains. OBJECTIVES: We aimed to apply next generation sequencing to describe transmission in hospital and compare with methods based on routinely-collected data. METHODS: All influenza samples taken through routine care from patients at University College London Hospitals NHS Foundation Trust (September 2012 to March 2014) were included. We conducted Illumina sequencing and identified genetic clusters. We compared nosocomial transmission estimates defined using classical methods (based on time from admission to sample) and genetic clustering. We identified pairs of cases with space-time links and assessed genetic relatedness. RESULTS: We sequenced influenza sampled from 214 patients. There were 180 unique genetic strains, 16 (8.8%) of which seeded a new transmission chain. Nosocomial transmission was indicated for 32 (15.0%) cases using the classical definition and 34 (15.8%) based on genetic clustering. Of the 50 patients in a genetic cluster, 11 (22.0%) had known space-time links with other cases in the same cluster. Genetic distances between pairs of cases with space-time links were lower than for pairs without spatial links (P < .001). CONCLUSIONS: Genetic data confirmed that nosocomial transmission contributes significantly to the hospital burden of influenza and elucidated transmission chains. Prospective next generation sequencing could support outbreak investigations and monitor the impact of infection and control measures.

Fire-mediated habitat change regulates woodland bird species and functional group occurrence.

In an era characterized by recurrent large wildfires in many parts of the globe, there is a critical need to understand how animal species respond to fires, the rates at which populations can recover, and the functional changes fires may cause. Using quantified changes in habitat parameters over a ~400-yr post-fire chronosequence in an obligate-seeding Australian eucalypt woodland, we build and test predictions of how birds, as individual species and aggregated into functional groups according to their use of specific habitat resources, respond to time since fire. Individual bird species exhibited four generalized response types to time since fire: incline, decline, delayed, and bell. All significant relationships between bird functional group richness or abundance and time since fire were consistent with predictions based on known time-since-fire-associated changes in habitat features putatively important for these bird groups. Consequently, we argue that the bird community is responding to post-fire successional changes in habitat as per the habitat accommodation model, rather than to time since fire per se, and that our functional framework will be of value in predicting bird responses to future disturbances in this and other obligate-seeder forest and woodland ecosystems. Most bird species and functional groups that were affected by time since fire were associated with long-unburned woodlands. In the context of recent large, stand-replacement wildfires that have affected a substantial proportion of obligate-seeder eucalypt woodlands, and the multi-century timescales over which post-fire succession occurs, it would appear preferable from a bird conservation perspective if fires initiating loss of currently long-unburned woodlands were minimized. Once long-unburned woodlands are transformed by fire into recently burned woodlands, there is limited scope for alternative management interventions to accelerate the rate of habitat development after fire, or supplement the resources formerly provided to birds by long-unburned woodlands, with the limited exception of augmenting hollow availability for key hollow-nesting species.

Modeling variability in the fire response of an endangered bird to improve fire-management.

Conservation managers regularly burn vegetation to regenerate habitat for fire-dependent species. When determining the time since fire at which to burn, managers model change in a species' occurrence over time, post-fire (fire-response curve) and identify the time since fire associated with decline in occurrence. However, where species exhibit variability in their fire response across space, using a single fire-response curve to determine the timing of burns may lead to burning habitat at an inappropriate time since fire. We tested if elevation, local topography, soil properties, vegetation type or evapotranspiration affect the fire response of the endangered Mallee Emu-wren Stipiturus mallee and its hummock-grass habitat Triodia scariosa in southeastern Australia (n = 217). Previous work on the Mallee Emu-wren found a unimodal fire response with decline in occurrence at ~30-50 yr since fire and a time window of occurrence of ~30 yr. We found that time since fire and elevation interact to affect the Mallee Emu-wren fire response. At high elevations (55-98 m), Mallee Emu-wrens declined in occurrence at ~50 yr since fire, with a time window of occurrence of 20-40 yr. However, at low elevations (28-55 m), Mallee Emu-wrens showed no decline in occurrence with increasing time since fire with a time window of occurrence of up to 107 yr. Extent cover of Tall T. scariosa showed similar patterns to the Mallee Emu-wren, indicating that vegetation structure is a likely driver of variability in the Mallee Emu-wren fire response. We speculate that the effect of low elevation is mediated by increased soil nutrient and water availability for key plants. We used our findings to map the appropriate time since fire at which to burn to regenerate habitat for the Mallee Emu-wren across the study region. We recommend no burning for regeneration across one-third of potential habitat, because the Mallee Emu-wren showed no decline in occurrence in these areas. We recommend managers model variability in species' fire responses across space to improve the timing of burns for regeneration.

Cassowary casques act as thermal windows.

Many ideas have been put forward for the adaptive value of the cassowary casque; and yet, its purpose remains speculative. Homeothermic animals elevate body temperature through metabolic heat production. Heat gain must be offset by heat loss to maintain internal temperatures within a range for optimal performance. Living in a tropical climate, cassowaries, being large bodied, dark feathered birds, are under thermal pressure to offload heat. We tested the original hypothesis that the casque acts as a thermal window. With infrared thermographic analyses of living cassowaries over an expansive range of ambient temperatures, we provide evidence that the casque acts as a thermal radiator, offloading heat at high temperatures and restricting heat loss at low temperatures. Interestingly, at intermediate temperatures, the casque appears thermally heterogeneous, with the posterior of the casque heating up before the front half. These findings might have implications for the function of similar structures in avian and non-avian dinosaurs.

Genome-Wide Sequence Analysis of Kaposi Sarcoma-Associated Herpesvirus Shows Diversification Driven by Recombination.

Background: Kaposi sarcoma-associated herpesvirus (KSHV) establishes lifelong infection in the human host and has been associated with a variety of malignancies. KSHV displays striking geographic variation in prevalence, which is highest in sub-Saharan Africa. The current KSHV genome sequences available are all tumor cell line-derived or primary tumor-associated viruses, which have provided valuable insights into KSHV genetic diversity. Methods: Here, we sequenced 45 KSHV genomes from a Ugandan population cohort in which KSHV is endemic; these are the only genome sequences obtained from nondiseased individuals and of KSHV DNA isolated from saliva. Results: Population structure analysis, along with the 25 published genome sequences from other parts of the world, showed whole-genome variation, separating sequences and variation within the central genome contributing to clustering of genomes by geography. We reveal new evidence for the presence of intragenic recombination and multiple recombination events contributing to the divergence of genomes into at least 5 distinct types. Discussion: This study shows that large-scale genome-wide sequencing from clinical and epidemiological samples is necessary to capture the full extent of genetic diversity of KSHV, including recombination, and provides evidence to suggest a revision of KSHV genotype nomenclature.

Does foliage metal accumulation influence plant-insect interactions? A field study of two sympatric tree metallophytes.

Gossia (Myrtaceae) is a highly restricted tree genus most speciose in New Caledonia and eastern Australia. The latter group accumulates above-normal foliar manganese (Mn) concentrations, with some individuals exhibiting the rare Mn-hyperaccumulative trait. Whether foliar metals contribute to chemical defence has been addressed via numerous feeding experiments and very few field studies. This investigation exploited specifically different insect activities on the foliage of sympatric Gossia grayi (N.Snow & Guymer) and Gossia shepherdii (F.Muell.) N.Snow & Guymer, endemic to north-eastern Australia, to test for direct and indirect effects of foliar Mn enrichment on plant-insect interactions. Leaf organic and inorganic chemistries, specific weight, surface damage, gall infestation and occupancy were quantified. Discovery that both species are Mn hyperaccumulators augments the world listing by 5-7%. Highly elevated gall-Mn concentrations coupled with negligible gall parasitisation suggested chemical fortification and adaptation by the host insect - a Cecidomyiidae fly. Linear mixed modelling (LMM) showed differences in leaf Mn, phenolics, toughness and surface damage across tree species and leaf age. There was no direct relationship between leaf Mn and insect impact. However, LMM did resolve indirect effects, i.e. between insect impact and certain foliar elements, consistent with nutritional dynamics in a physiologically novel plant system where Mn is vastly overaccumulated.

Genome-wide evolutionary dynamics of influenza B viruses on a global scale.

The global-scale epidemiology and genome-wide evolutionary dynamics of influenza B remain poorly understood compared with influenza A viruses. We compiled a spatio-temporally comprehensive dataset of influenza B viruses, comprising over 2,500 genomes sampled worldwide between 1987 and 2015, including 382 newly-sequenced genomes that fill substantial gaps in previous molecular surveillance studies. Our contributed data increase the number of available influenza B virus genomes in Europe, Africa and Central Asia, improving the global context to study influenza B viruses. We reveal Yamagata-lineage diversity results from co-circulation of two antigenically-distinct groups that also segregate genetically across the entire genome, without evidence of intra-lineage reassortment. In contrast, Victoria-lineage diversity stems from geographic segregation of different genetic clades, with variability in the degree of geographic spread among clades. Differences between the lineages are reflected in their antigenic dynamics, as Yamagata-lineage viruses show alternating dominance between antigenic groups, while Victoria-lineage viruses show antigenic drift of a single lineage. Structural mapping of amino acid substitutions on trunk branches of influenza B gene phylogenies further supports these antigenic differences and highlights two potential mechanisms of adaptation for polymerase activity. Our study provides new insights into the epidemiological and molecular processes shaping influenza B virus evolution globally.

Correction to "Discovery of Fevipiprant (NVP-QAW039), a Potent and Selective DP2 Receptor Antagonist for Treatment of Asthma".

[This corrects the article DOI: 10.1021/acsmedchemlett.7b00157.].

Discovery of Fevipiprant (NVP-QAW039), a Potent and Selective DP2 Receptor Antagonist for Treatment of Asthma.

Further optimization of an initial DP2 receptor antagonist clinical candidate NVP-QAV680 led to the discovery of a follow-up molecule 2-(2-methyl-1-(4-(methylsulfonyl)-2-(trifluoromethyl)benzyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acetic acid (compound 11, NVP-QAW039, fevipiprant), which exhibits improved potency on human eosinophils and Th2 cells, together with a longer receptor residence time, and is currently in clinical trials for severe asthma.

The global antigenic diversity of swine influenza A viruses.

Swine influenza presents a substantial disease burden for pig populations worldwide and poses a potential pandemic threat to humans. There is considerable diversity in both H1 and H3 influenza viruses circulating in swine due to the frequent introductions of viruses from humans and birds coupled with geographic segregation of global swine populations. Much of this diversity is characterized genetically but the antigenic diversity of these viruses is poorly understood. Critically, the antigenic diversity shapes the risk profile of swine influenza viruses in terms of their epizootic and pandemic potential. Here, using the most comprehensive set of swine influenza virus antigenic data compiled to date, we quantify the antigenic diversity of swine influenza viruses on a multi-continental scale. The substantial antigenic diversity of recently circulating viruses in different parts of the world adds complexity to the risk profiles for the movement of swine and the potential for swine-derived infections in humans.

Historical Maps from Modern Images: Using Remote Sensing to Model and Map Century-Long Vegetation Change in a Fire-Prone Region.

Understanding the age structure of vegetation is important for effective land management, especially in fire-prone landscapes where the effects of fire can persist for decades and centuries. In many parts of the world, such information is limited due to an inability to map disturbance histories before the availability of satellite images (~1972). Here, we describe a method for creating a spatial model of the age structure of canopy species that established pre-1972. We built predictive neural network models based on remotely sensed data and ecological field survey data. These models determined the relationship between sites of known fire age and remotely sensed data. The predictive model was applied across a 104,000 km(2) study region in semi-arid Australia to create a spatial model of vegetation age structure, which is primarily the result of stand-replacing fires which occurred before 1972. An assessment of the predictive capacity of the model using independent validation data showed a significant correlation (rs = 0.64) between predicted and known age at test sites. Application of the model provides valuable insights into the distribution of vegetation age-classes and fire history in the study region. This is a relatively straightforward method which uses widely available data sources that can be applied in other regions to predict age-class distribution beyond the limits imposed by satellite imagery.

Fevipiprant (QAW039), a Slowly Dissociating CRTh2 Antagonist with the Potential for Improved Clinical Efficacy.

Here we describe the pharmacologic properties of a series of clinically relevant chemoattractant receptor-homologous molecules expressed on T-helper type 2 (CRTh2) receptor antagonists, including fevipiprant (NVP-QAW039 or QAW039), which is currently in development for the treatment of allergic diseases. [(3)H]-QAW039 displayed high affinity for the human CRTh2 receptor (1.14 ± 0.44 nM) expressed in Chinese hamster ovary cells, the binding being reversible and competitive with the native agonist prostaglandin D2(PGD2). The binding kinetics of QAW039 determined directly using [(3)H]-QAW039 revealed mean kinetic on (kon) and off (koff) values for QAW039 of 4.5 × 10(7)M(-1)min(-1)and 0.048 minute(-1), respectively. Importantly, thekoffof QAW039 (half-life = 14.4 minutes) was >7-fold slower than the slowest reference compound tested, AZD-1981. In functional studies, QAW039 behaved as an insurmountable antagonist of PGD2-stimulated [(35)S]-GTPγS activation, and its effects were not fully reversed by increasing concentrations of PGD2after an initial 15-minute incubation period. This behavior is consistent with its relatively slow dissociation from the human CRTh2 receptor. In contrast for the other ligands tested this time-dependent effect on maximal stimulation was fully reversed by the 15-minute time point, whereas QAW039's effects persisted for >180 minutes. All CRTh2 antagonists tested inhibited PGD2-stimulated human eosinophil shape change, but importantly QAW039 retained its potency in the whole-blood shape-change assay relative to the isolated shape change assay, potentially reflective of its relatively slower off rate from the CRTh2 receptor. QAW039 was also a potent inhibitor of PGD2-induced cytokine release in human Th2 cells. Slow CRTh2 antagonist dissociation could provide increased receptor coverage in the face of pathologic PGD2concentrations, which may be clinically relevant.

Rapid outbreak sequencing of Ebola virus in Sierra Leone identifies transmission chains linked to sporadic cases.

To end the largest known outbreak of Ebola virus disease (EVD) in West Africa and to prevent new transmissions, rapid epidemiological tracing of cases and contacts was required. The ability to quickly identify unknown sources and chains of transmission is key to ending the EVD epidemic and of even greater importance in the context of recent reports of Ebola virus (EBOV) persistence in survivors. Phylogenetic analysis of complete EBOV genomes can provide important information on the source of any new infection. A local deep sequencing facility was established at the Mateneh Ebola Treatment Centre in central Sierra Leone. The facility included all wetlab and computational resources to rapidly process EBOV diagnostic samples into full genome sequences. We produced 554 EBOV genomes from EVD cases across Sierra Leone. These genomes provided a detailed description of EBOV evolution and facilitated phylogenetic tracking of new EVD cases. Importantly, we show that linked genomic and epidemiological data can not only support contact tracing but also identify unconventional transmission chains involving body fluids, including semen. Rapid EBOV genome sequencing, when linked to epidemiological information and a comprehensive database of virus sequences across the outbreak, provided a powerful tool for public health epidemic control efforts.

Molecular Epidemiology and Evolution of Influenza Viruses Circulating within European Swine between 2009 and 2013.

UNLABELLED: The emergence in humans of the A(H1N1)pdm09 influenza virus, a complex reassortant virus of swine origin, highlighted the importance of worldwide influenza virus surveillance in swine. To date, large-scale surveillance studies have been reported for southern China and North America, but such data have not yet been described for Europe. We report the first large-scale genomic characterization of 290 swine influenza viruses collected from 14 European countries between 2009 and 2013. A total of 23 distinct genotypes were identified, with the 7 most common comprising 82% of the incidence. Contrasting epidemiological dynamics were observed for two of these genotypes, H1huN2 and H3N2, with the former showing multiple long-lived geographically isolated lineages, while the latter had short-lived geographically diffuse lineages. At least 32 human-swine transmission events have resulted in A(H1N1)pdm09 becoming established at a mean frequency of 8% across European countries. Notably, swine in the United Kingdom have largely had a replacement of the endemic Eurasian avian virus-like ("avian-like") genotypes with A(H1N1)pdm09-derived genotypes. The high number of reassortant genotypes observed in European swine, combined with the identification of a genotype similar to the A(H3N2)v genotype in North America, underlines the importance of continued swine surveillance in Europe for the purposes of maintaining public health. This report further reveals that the emergences and drivers of virus evolution in swine differ at the global level. IMPORTANCE: The influenza A(H1N1)pdm09 virus contains a reassortant genome with segments derived from separate virus lineages that evolved in different regions of the world. In particular, its neuraminidase and matrix segments were derived from the Eurasian avian virus-like ("avian-like") lineage that emerged in European swine in the 1970s. However, while large-scale genomic characterization of swine has been reported for southern China and North America, no equivalent study has yet been reported for Europe. Surveillance of swine herds across Europe between 2009 and 2013 revealed that the A(H1N1)pdm09 virus is established in European swine, increasing the number of circulating lineages in the region and increasing the possibility of the emergence of a genotype with human pandemic potential. It also has implications for veterinary health, making prevention through vaccination more challenging. The identification of a genotype similar to the A(H3N2)v genotype, causing zoonoses at North American agricultural fairs, underlines the importance of continued genomic characterization in European swine.

Genome diversity of Epstein-Barr virus from multiple tumor types and normal infection.

UNLABELLED: Epstein-Barr virus (EBV) infects most of the world's population and is causally associated with several human cancers, but little is known about how EBV genetic variation might influence infection or EBV-associated disease. There are currently no published wild-type EBV genome sequences from a healthy individual and very few genomes from EBV-associated diseases. We have sequenced 71 geographically distinct EBV strains from cell lines, multiple types of primary tumor, and blood samples and the first EBV genome from the saliva of a healthy carrier. We show that the established genome map of EBV accurately represents all strains sequenced, but novel deletions are present in a few isolates. We have increased the number of type 2 EBV genomes sequenced from one to 12 and establish that the type 1/type 2 classification is a major feature of EBV genome variation, defined almost exclusively by variation of EBNA2 and EBNA3 genes, but geographic variation is also present. Single nucleotide polymorphism (SNP) density varies substantially across all known open reading frames and is highest in latency-associated genes. Some T-cell epitope sequences in EBNA3 genes show extensive variation across strains, and we identify codons under positive selection, both important considerations for the development of vaccines and T-cell therapy. We also provide new evidence for recombination between strains, which provides a further mechanism for the generation of diversity. Our results provide the first global view of EBV sequence variation and demonstrate an effective method for sequencing large numbers of genomes to further understand the genetics of EBV infection. IMPORTANCE: Most people in the world are infected by Epstein-Barr virus (EBV), and it causes several human diseases, which occur at very different rates in different parts of the world and are linked to host immune system variation. Natural variation in EBV DNA sequence may be important for normal infection and for causing disease. Here we used rapid, cost-effective sequencing to determine 71 new EBV sequences from different sample types and locations worldwide. We showed geographic variation in EBV genomes and identified the most variable parts of the genome. We identified protein sequences that seem to have been selected by the host immune system and detected variability in known immune epitopes. This gives the first overview of EBV genome variation, important for designing vaccines and immune therapy for EBV, and provides techniques to investigate relationships between viral sequence variation and EBV-associated diseases.

Accumulation of human-adapting mutations during circulation of A(H1N1)pdm09 influenza virus in humans in the United Kingdom.

UNLABELLED: The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the United Kingdom, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second- and third-wave viruses replicated more rapidly in human airway epithelial (HAE) cells than did the first-wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second- and third-wave viruses induced less alpha interferon in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second- and third-wave viruses. Recombinant viruses with the third-wave NS gene induced less interferon in human cells, but this alone did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third-wave viruses caused increased binding to α-2,6-sialic acid and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third-wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness, which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza virus during successive waves. IMPORTANCE: Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown, but the numbers of severe cases increased during successive waves of human infection in the United Kingdom. To determine the causes of this variation, we studied genetic changes in virus isolates from individual hospitalized patients. There were no consistent differences between these viruses and those circulating in the community, but we found multiple evolutionary changes that in combination over time increased the virus's ability to infect human cells. These adaptations may explain the remarkable ability of A(H1N1)pdm09 virus to continue to circulate despite widespread immunity and the apparent increase in severity of influenza over successive waves of infection.

Generation and characterization of influenza A viruses with altered polymerase fidelity.

Genetic diversity of influenza A viruses (IAV) acquired through the error-prone RNA-dependent RNA polymerase (RdRP) or through genetic reassortment enables perpetuation of IAV in humans through epidemics or pandemics. Here, to assess the biological significance of genetic diversity acquired through RdRP, we characterize an IAV fidelity variant derived from passaging a seasonal H3N2 virus in the presence of ribavirin, a purine analogue that increases guanosine-to-adenosine mutations. We demonstrate that a single PB1-V43I mutation increases selectivity to guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses. The H5N1 PB1-V43I-recombinant virus replicates to comparable titres as the wild-type virus in vitro or in the mouse lungs. However, a decrease in viral population diversity at day 3 post inoculation is associated with a tenfold reduced lethality and neurotropism in mice. Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis.

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.

Respiratory tract samples, viral load, and genome fraction yield in patients with Middle East respiratory syndrome.

BACKGROUND: Analysis of clinical samples from patients with new viral infections is critical to confirm the diagnosis, to specify the viral load, and to sequence data necessary for characterizing the viral kinetics, transmission, and evolution. We analyzed samples from 112 patients infected with the recently discovered Middle East respiratory syndrome coronavirus (MERS-CoV). METHODS: Respiratory tract samples from cases of MERS-CoV infection confirmed by polymerase chain reaction (PCR) were investigated to determine the MERS-CoV load and fraction of the MERS-CoV genome. These values were analyzed to determine associations with clinical sample type. RESULTS: Samples from 112 individuals in which MERS-CoV was detected by PCR were analyzed, of which 13 were sputum samples, 64 were nasopharyngeal swab specimens, 30 were tracheal aspirates, and 3 were bronchoalveolar lavage specimens; 2 samples were of unknown origin. Tracheal aspirates yielded significantly higher MERS-CoV loads, compared with nasopharyngeal swab specimens (P = .005) and sputum specimens (P = .0001). Tracheal aspirates had viral loads similar to those in bronchoalveolar lavage samples (P = .3079). Bronchoalveolar lavage samples and tracheal aspirates had significantly higher genome fraction than nasopharyngeal swab specimens (P = .0095 and P = .0002, respectively) and sputum samples (P = .0009 and P = .0001, respectively). The genome yield from tracheal aspirates and bronchoalveolar lavage samples were similar (P = .1174). CONCLUSIONS: Lower respiratory tract samples yield significantly higher MERS-CoV loads and genome fractions than upper respiratory tract samples.