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HPFS (n = ), ± , ± , ± , Morales E, Bustamante M, Gonzalez JR, Guxens M, Torrent M, Mendez M. For samples requiring extra coverage, the Ion Torrent Personal Genome Machine A in VP3, N in VP1, N in 2A, K in 3D, and G at the 2B/2C.

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However, in EV-D68 was responsible for more than 1, disease cases in North America, including severe respiratory illness in children and acute flaccid myelitis, raising concerns about its potential impact on public health. Despite the emergence of EV-D68, a lack of full-length genome sequences means that little is known about the molecular evolution of this virus within a single geographic locality during a single outbreak.

Here, we doubled the number of publicly available complete genome sequences of EV-D68 by performing high-throughput next-generation sequencing, characterized the evolutionary history of this outbreak in detail, identified a recombination event, and investigated whether there was any correlation between the demographic and clinical characteristics of the patients and the viral variant that infected them.

Overall, these results will help inform the design of intervention strategies for EV-D All EVs are characterized by a single positive-strand RNA genome of approximately 7, nucleotides nt in length. EV-D68 was first isolated from hospitalized children with pneumonia and bronchiolitis in California in 8 , and rhinovirus 87 was later reclassified as EV-D68 based on phylogenetic analysis, cross-neutralization, and acid sensitivity 9 , — EV-D68 was detected sporadically from the s through the early s 12 , — However, an increased number of infections by EV-D68 has been reported worldwide during the past decade 15 , 16 , 18 , — Almost all documented EV-D68 cases during this time were associated with acute respiratory infections, ranging from mild upper respiratory tract illness and asthma to severe bronchitis and pneumonia, with the exception of four isolated cases associated with neurological syndromes, notably, acute flaccid myelitis AFM , in which viruses were isolated from the cerebrospinal fluid or nasopharyngeal swabs 12 , 31 , The majority of cases were associated with severe respiratory illness in children 36 , 37 leading to hospitalization.

Previous studies have revealed the presence of three major clades of EV-D68, designated A, B and C, which have circulated or cocirculated during different time periods in different geographic regions 18 , Along with high rates of nucleotide substitution, recombination is an important way in which genetic diversity can be generated in enteroviruses However, no recombination events have yet been reported in EV-D68, in part reflecting a lack of full-length genome sequences for analysis There is growing interest in understanding the epidemiology of EV-D68 in the United States, particularly given its association with severe disease outcomes during the outbreak.

Indeed, little is known about the molecular evolution of the virus during a single outbreak, nor whether differences in clinical manifestation are associated with specific genetic variants. The most important limitation here has been the lack of complete genome sequences of EV-D68 in the public domain, even though such information will assist the design of effective intervention and prevention strategies and help formulate modalities of future treatments. To help determine the evolution of EV-D68 within a single geographic region during a single outbreak, we performed full-length genome sequencing of viruses sampled from children seen at Children's Mercy Hospital—the first hospital reporting the EV-D68 outbreak in —in Kansas City, MO, during August to September As well as determining the extent and pattern of viral genetic diversity, we screened these genomic data for recombination and assessed whether there was any association between genetic variation and specific demographic and clinical features of the infected patients.

Data were retrospectively obtained from chart abstraction and entered into a standardized data collection instrument. A single-term, previously healthy infant with EV-D68 was enrolled in an infant birth cohort based at Vanderbilt University Medical Center and was included as a preemergent isolate in a region geographically close to Kansas City.

This is a population-based birth cohort, and respiratory illness surveillance was performed every 2 weeks during the winter season. All information was prospectively collected, and parents gave their informed consent for study inclusion Extraction of viral RNA was performed at the J.

In brief, specimen lysis was performed in Qiagen buffer AVL in a well deep-well plate. Lysate was transferred to a ZR96 spin plate Zymo , and samples were processed according to the manufacturer's protocol. Three independent approaches were taken when performing PCR amplification of the viral genome.

A large L 6. The resulting contigs were searched against custom, full-length EV-D68 nucleotide databases to identify the closest reference sequence. At loci where both Illumina and Ion Torrent sequence data agreed on a variation compared with the reference sequence, it was updated to reflect the difference.

VIGOR was used to predict genes, perform alignments, ensure the fidelity of open reading frames, associate nucleotide polymorphisms with amino acid changes, and detect any potential sequencing errors. The annotation was subjected to manual inspection and quality control before submission to GenBank. In total, we utilized two global data sets of EV-D a complete-genome data set 59 JCVI-sequenced sequences and 51 background sequences and a complete-VP1-gene data set 59 JCVI-sequenced sequences and background sequences.

The robustness of the ML tree was assessed by bootstrap analyses of 1, pseudoreplicates and by comparison with the topologies sampled in the Bayesian analysis. Such changes in geographic state are indicative of independent viral introductions into Kansas City. To confirm these putative recombination events, we utilized a smaller data set including the recombinant and the parental strains determined above, employing similarity plots and Bootscan analysis as implemented in Simplot version 3. The midpoint of the breakpoint region was used to partition the sequence alignment for separate phylogenetic inferences.

Demographic and clinical information was available for 57 outbreak samples from Kansas City. Similarly, each patient was classified as either positive or negative for each clinical symptom individually. The clinical symptoms analyzed were i presence in pediatric ICU, ii medical history of asthma or recurrent wheezing, and iii requirement for ventilation.

The strength of association between the phenotypic features described above and the EV-D68 phylogeny was determined using two phylogeny-trait association statistics, the parsimony score PS and the association index AI tests, both of which were implemented in the Bayesian tip association significance testing BaTS program A null distribution of these statistics was determined using the posterior distribution of trees obtained from the MrBayes analysis described above. The general pattern of primer sites and the locations of primer targets in EV-D68 genomes are shown in Fig.

To achieve complete genome sequencing, three approaches were attempted from respiratory samples: i full-length genome with poly A tail FL-A , ii full-length genome without the poly A tail FL , and iii two overlapping amplicons—large L and small S Fig. Representative PCR products for each method are shown in Fig. In total, we obtained 59 complete genome sequences of EV-D68, including 57 outbreak samples from Kansas City, one preoutbreak sample from Nashville, and one historical sample from ATCC.

High-throughput sequencing of the complete genome of EV-D A Schematic representation of the different approaches used to sequence the complete EV-D68 genome. Sizes of molecular markers in base pairs are indicated on the left side of the gel. Deep sequence analysis revealed an average of 4. Together, these new complete genome sequences doubled the number available on GenBank. Phylogenetic analyses of the EV-D68 VP1 gene and complete genome sequences revealed that the viruses that circulated in Kansas City during the outbreak did not form a single monophyletic group, such that EV-D68 was clearly introduced multiple times into the city Fig.

Clades and subclades are indicated by colors and described in the trees. Sequences reported by this study are shown in red and described in the key. Sequences generated in this study are marked by red squares in the tree.

Sequences collected from outside the United States have taxon names in blue. The scale bars represent the numbers of nucleotide substitutions per site. All 57 outbreak samples from Kansas City fell into subclade B1. Within this subclade, it was notable that the Kansas City viruses fell into two monophyletic groups and were interspersed among those viruses sampled from different states in the United States New York, California, and Colorado and from different countries Canada or France , indicating frequent gene flow between populations Fig.

However, because of low bootstrap support values and frequent polytomies, it is difficult to define exact monophyletic groups of viruses from Kansas City on global VP1 phylogeny. Therefore, we estimated the number of independent viral introductions into the Kansas City population using a parsimony reconstruction of geographic transitions, utilizing 5, subsampled BMCMC topologies to reflect topological uncertainty. The large and well-supported monophyletic group of viruses in subclade B1 at the top of Fig.

Again, this phylogenetic pattern is indicative of both the national and international movement of viruses. Seven Kansas City sequences did not fall in this cluster; four were closely related to each other, while another sequence grouped with a virus from Canada.

The remaining two Kansas City sequences formed separate branches in the tree, suggesting that they also represent independent introductions into the region. The remaining subclade B1 viruses are preoutbreak samples located near the root of the B1 cluster, including four U. In addition, only five U. Sequence US. Finally, there are two Fermon strain sequences in our analysis, one downloaded from GenBank and the prototype Fermon strain that we purchased from ATCC and sequenced.

These two sequences clustered near the root, reflecting its sampling date in the s Fig. The phylogeny of complete EV-D68 genome sequences presented the same picture of three major clades circulating worldwide and multiple viral introductions to Kansas City during the outbreak Fig. The nucleotide similarities among the clades ranged from VP1 is the most variable gene across the genome as a whole, displaying Phylogenetic trees of the complete genome and individual genes of EV-D Sequences in different clades are colored as described in the key.

Phylogenetic trees inferred for different regions of the EV-D68 genome are indicated in black. Phylogenetic trees were rooted using the oldest EV-D68 sequence the Fermon strain available on GenBank, and scale bars represent the numbers of nucleotide substitutions per site.

We found no overt evidence of homologous recombination in the VP1 data set. However, a single recombinant virus—strain US. Although the Bootscan analysis provided evidence for a second recombination event in the 3D gene region, this lacked statistical support. Hence, these data suggest that there was an intersubclade recombination event between B2 and B1.

In contrast to subclade B1, which is the main U. Among the latter, one is the confirmed recombinant, and another is the potential parental strain, US. Recombination analyses of the full genome of EV-D A Bootscan analysis. A sliding window of nt was utilized step size of 10 nt , with neighbor-joining phylogenetic trees with bootstrap replicates inferred in each case.

Bootstrap values supporting the clustering of query sequence i. B Schematic diagram of the EV-D68 genome. C Phylogenies inferred for nonrecombinant fragments identified by Simplot and Bootscan A. The putative recombinant strain is marked by the black square in the trees. Clades are indicated by the colors described in the key.

Phylogenetic trees were midpoint rooted for clarity only. We performed two phylogeny-trait association statistics AI and PS to identify whether EV-D68 outbreak strains might exhibit some phylogenetic clustering by disease severity. Notably, however, we found no significant association of age distribution, gender ratio, or clinical symptomatology with viral genetic variation i. Hence, phylogenetic clustering by disease phenotype was not greater than that expected by chance alone.

Results of the phylogeny-trait association tests for particular demographic and clinical characteristics of EV-D68 patients within Kansas City, MO, We developed a high-throughput method to sequence complete genomes of EV-D68, from which we were able to obtain 59 from the major outbreak, including 57 from Kansas City. This doubles the number of publicly available genome sequences of EV-D68 and allowed us to identify both multiple introduction events into a single community and multiple cocirculating lineages worldwide, although there was no association between phylogenetic history and a range of demographic or clinical features.

Notably, we also identified the first recombination event in EV-D Until now, only limited full-length genome sequences of EV-D68 from the EV-D68 outbreak in the United States have been available in the public domain, with most coming from clinical isolates.

Our phylogenetic analyses are notable in that they suggest a relatively complex molecular evolution of EV-D68 during the outbreak. Before this outbreak, EV-D68 was rarely reported and was associated with mild respiratory illness, although small outbreaks had been documented since 15 , 16 , 18 , — Previous studies showed that the A, B, and C clades circulated or cocirculated during different time periods in different geographic regions 15 , 16 , 18 , 19 , 21 , — 32 , We further defined subclades A1, A2, B1, and B2.

In line with our subclade definition, subclades A1 and B2 were endemic and found in many countries before the outbreak, including Thailand during to 26 , the United Kingdom during to 27 , China during to 24 , Philippines from to 21 , — 23 , and the Netherlands from to 15 , Clade C was relatively geographically restricted, being reported in Japan during to and in Italy during to 16 , 19 , During the outbreak, a novel subclade, B1, seems to have rapidly emerged and was dominant during the U.

During the same period, subclades A1 and B2 continued to be isolated in European countries 40 , 41 , 62 , — In addition, we found multiple viral introductions from other localities into the single locality of Kansas City during the U. Our study is noteworthy for the observation of recombination in EV-D Although recombination occurs frequently among members of the Enterovirus genus, with interspecies recombination documented between rhinovirus A and rhinovirus B and between EV-A and EV-B 33 , and intraspecies recombination within both EV-A71 and EV-B81 44 , 66 , it had not been previously definitely demonstrated to occur in EV-D In recombination events in other enteroviruses, most breakpoints have been documented between the structural and nonstructural regions, or within the nonstructural region 44 , The recombinant strain identified here, US.

In turn, the occurrence of recombination indicates that multiple distinct strains cocirculated at the beginning of the outbreak, as is also suggested by our phylogenetic analysis. One of the most striking features of the outbreak was that most EV-D68 infections were associated with severe respiratory diseases in children 36 , 40 , 41 , 60 , — 65 , However, we found no association between age, gender, or clinical symptoms and different viral subclades in our Kansas City population. Hence, whether sequence data can predict the clinical outcome is still unclear, and our sample size was relatively small.

One finding suggestive of an association between viral sequence diversity and clinical features is that AFM-associated strains from the United States during the outbreak were all members of the novel outbreak subclade B1 38 , although we were unable to identify viral genetic signatures of disease severity. Indeed, there are a number of features that argue against a strong strain basis to virulence.

First, of the four cases of neurological conditions before the outbreak, two cases from Kenya strain HEV in and strain HEV in were associated with viruses from subclade A1, the endemic subclade 31 , while sequence information concerning the two U. Second, although all U. However, G was present in all the clades studied here and the polyprotein mutations were B1 specific, not AFM specific, as they were also found in non-AFM patients.

Although we considered only a single city in the United States during the outbreak, these results are likely to be applicable to the spread of EV-D68 in other modern and highly mobile populations. For an improved understanding of the factors determining possible spatiotemporal differences in EV-D68 infection and transmission, continuous global monitoring of the clinical and molecular epidemiology of EV-D68 by representative surveillance systems should clearly be a public health priority.

The content is solely the responsibility of the authors and does not represent official views of the National Institutes of Health. J Virol. Published online Jan Prepublished online Dec 9. Chappell , e Tina V. Hartert , f Edward C. Holmes , g and Suman R. Das a. Jennifer E. Rebecca A.

Halpin a Infectious Diseases Group, J. Timothy B. Stockwell a Infectious Diseases Group, J. James D. Tina V. Edward C. Suman R. Das a Infectious Diseases Group, J. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Address correspondence to Suman R. Das, gro. Molecular evolution and intraclade recombination of enterovirus D68 during the outbreak in the United States. J Virol — Received Sep 18; Accepted Nov All Rights Reserved. This article has been cited by other articles in PMC.

September 17, , PM Most reported problems are fixed by this: It is simple to remember that you have to open Steam before opening the launcher. All further requests for support in this thread will be deleted, please create a new thread in the correct forum.

No more wild AI charging every unit from across the battlefield. Very fun. One thing that I have noticed - the AI never quits even when things are hopeless. For instance if they have a 10 stack army reinforced by a 5 stack army and I wipe out the 10 stack army before the 5 stack makes it fully onto the battlefield, the 5 stack will still attack me even though it has no chance.

It'd be cool if the AI said 'no thanks' and just withdrew as soon as things were clearly hopeless. Not sure if this is possible or not. This is hard coded I believe, I cannot make the AI to withdraw. Tried some morale tweaks, no luck. But overall it is quite ruthless I think. Not sure how exactly you're able to do this but I will say it has be interested in starting to learn to mod a bit.

Did you ever see my tweet about setting up a paypal donation link? The deployment of the fences blocks the cavalry on the rear. I tried 2 custom battles, with the same results. Here a screen: Spoiler Alert, click show to read:. It really sounded like it was Quickly did a reboot.

Apart from that , I'm very excited to see the 8. I've been waiting for it for a long time , lurking around the Darthmod thread. Thank you so much for trying so hard to salvage Empire Total War. Now it's playtime. Only gripe is x40 unit campaigns have 2 turns per year. AI performance in Campaign is much better, battle AI as well. CAI seemed improved even from beta Sweden had 1.

Noted my units don't take firing position on the wall after switching from melee mode. I have to move them couple yards, then they get into firing position. Sent from my N using Tapatalk 2. Last edited by F 32; September 17, at PM. The Darthmod series is amazing and I absolutely love them. Well done!!

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