to ascertain the etiologic agent causing an outbreak of febrile illness with symptoms similar to chikungunya fever , we selected 3 sites in chiapas state , mexico , for sampling : tapachula , la libertad , and ciudad hidalgo ( figure 1 , panel a ) .
after patients informed consent was obtained , blood samples were collected from persons whose condition met the following case definition for possible chikungunya fever : acute onset of fever > 38.5c , accompanied by severe arthralgia not explained by other medical conditions ( 6 ) .
samples from tapachula were collected from patients who sought treatment at the centro regional de investigacion en salud publica , whereas in la libertad and ciudad hidalgo , researchers surveyed houses to identify potential case - patients . in total , 119 blood samples were collected , and serum was isolated by centrifugation .
six samples were stored in magnapure lc buffer ( roche , nutley , nj , usa ) , which inactivates virus particles but preserves the genomic rna .
map of mexico showing the 3 sites where serum samples were obtained to test for chikungunya virus in chiapas , mexico , 2014 : tapachula , la libertad , and ciudad hidalgo .  
viral rna was extracted from serum samples using the zr-96 viral rna kit ( zymo research , orange , ca , usa ) according to the manufacturer s protocol .
one - step quantitative reverse transcription pcr ( qrt - pcr ) ( 7 ) was performed by using the taqman rna - to - ct 1-step kit ( applied biosystems , san francisco , ca , usa ) .
standard plaque assays were performed for the samples positive by qrt - pcr with vero cells .
anti - chikv igm - capture enzyme - linked immunosorbent assays ( elisas ) ( 8) were performed by using a chimeric eilat - chikv ( 9 ) that contained the nonstructural proteins from eilat virus and structural proteins from chikv , resulting in a structure indistinguishable from that of chikv .
a sample was considered to be chikv negative if the sample was not positive by qrt - pcr or igm elisa .
viral rna from 5 serum samples was sent for illumina deep sequencing and assembled by using the virus - specific hive - hexagon algorithm ( 10 ) and the ngen module in lasergene suite version 10 ( bioinformatics pioneer dnastar , inc . , madison , wi , usa ) .
single nucleotide polymorphisms were analyzed by using the sequencing profiling tool in the hive suite of programs ( 10 ) .
sequences were aligned in seaview ( 11 ) by using translated proteins for the open reading frames and using nucleotides for the untranslated genome regions , and all gaps were removed .
bayesian phylogenetic inference was performed by using the general time reversible plus invariant sites plus gamma distribution 4 model in mrbayes ( 12 ) with 1.5 million iterations to reach congruence .
partial genome sequencing of the e2 and e1 glycoproteins was performed by using traditional sanger methods on pcr amplicons on an additional 8 samples .
over 100 serum samples were obtained from persons seeking treatment for chikungunya fever  like illness during october  december 2014 in 3 locations in chiapas , mexico ( tapachula , la libertad , ciudad hidalgo ) ( figure 1 ) .
these samples were analyzed by qrt - pcr and igm - capture elisa to detect viremia and igm , respectively .
no overlap occurred between the samples that were positive for chikv by qrt - pcr or those positive by igm , demonstrating the importance of the humoral response to viral clearance . with few exceptions ,
viremia was detectable up to 3 days after fever onset ( figure 2 , panel a ) , after which most samples were igm positive .
all age groups were equally likely to be infected , as expected during infectious disease outbreaks involving a naive population ( 13 ) .  
a ) number of serum samples positive for chikungunya virus ( chikv ) by reverse transcription quantitative pcr ( qrt - pcr ) , for chikv igm by elisa , and negative for chikv by both methods , arranged according to day after fever onset .
b ) phylogenetic tree generated by bayesian analysis showing the relationship of the complete genomic sequences of 5 chikungunya virus isolates from mexico and representative sequences from the genbank library .
all nodes showed posterior probabilities of > 0.9 , except those indicated with a star .
plaque assays were performed to determine serum virus titers ( table ) ; 3 qrt - pcr samples from tapachula could not be assayed because of sample limitations .
mean viremia level was similar among the 3 sampled locations and ranged from < 2 log10 to 4.2 log10 pfu / ml .
* chikv , chikungunya virus ; qrt - pcr , quantitative reverse transcription pcr . 
six patient samples that were negative for chikv by qrt - pcr were unable to be used for plaque assays or elisas because they were stored in magnapure buffer ( roche , nutley , nj , usa ) .
five serum samples from diverse locations and collection dates were selected for illumina sequencing ( genbank accession nos .
the strains circulating in chiapas were most closely related to asian lineage strains first detected in the caribbean ( represented by a british virgin islands isolate ) and now presumed to be circulating in much of latin america ( figure 2 , panel b ) .
curiously , no novel mutations appear to have been fixed in the year since the british virgin islands isolate was collected .
although the genomic sequences confirmed that the circulating virus in chiapas belonged to the asian lineage , which is primarily transmitted by a. aegypti mosquitoes ( 1 ) , we nevertheless examined the sequences for mutations known to adapt chikv for transmission by a. albopictus mosquitoes . because both mosquito species are found in chiapas ( 14 ) , adaptation of the chikv strain circulating in mexico to a. albopictus mosquitoes could place temperate regions of the eastern united states and millions of naive persons at risk for infection .
none of the e2 or e1 substitutions previously reported to increase fitness in a. albopictus mosquitoes was observed in 8 additional samples analyzed by sanger sequencing ( genbank accession nos .
one sample had a nonsynonymous mutation , in comparison to the january 2014 british virgin islands isolate ( genbank accession no .
we found that 79% of febrile illness cases with polyarthralgia in chiapas state during late 2014 were caused by chikv .
our sequencing of chikv genomes confirmed spread of an asian lineage strain from the caribbean and suggested that although chikv has circulated in the americas since 2013 , no adaptive mutations have occurred .
however , continued screening for vector - adaptive mutations will be critical , especially now that strains of the ecsa lineage , which gave rise to the indian ocean lineage , have been introduced into brazil ( 4 ) .
 since chikungunya virus ( chikv ) was introduced into the americas in 2013 , its geographic distribution has rapidly expanded . 
 of 119 serum samples collected in 2014 from febrile patients in southern mexico , 79% were positive for chikv or igm against chikv . 
 sequencing results confirmed chikv strains closely related to caribbean isolates . 
The Study
Conclusions