we conducted a retrospective study by using samples in our biobank : 1 ) acute - phase serum and supernatant from ndcp collected during january 2007july 2016 ; 2 ) acute- or convalescent - phase serum and supernatant samples collected by private clinics for dengue diagnosis during the same period ; and 3 ) urine from dengue - negative patients and mosquito samples included in the denfree study conducted in cambodia during 20122013 ( technical appendix 1 ) . the use of samples was approved by the national ethics committee for health research of cambodia . aegypti female mosquitoes negative for dengue infection by quantitative real - time reverse transcription pcr ( qrt - pcr ) . in total , 2,400 serum samples and 173 supernatants from c6/36 cell cultures of serum from the dengue surveillance system , 270 urine samples from the denfree study , and 3,159 mosquitoes were tested for zika virus by qrt - pcr ( 10 ) . positive results were confirmed by using conventional rt - pcr targeting the nonstructural protein 5 gene ( 11 ) and the trioplex qrt - pcr kit ( 12 ) . dna products of conventional rt - pcr were sent for sequencing to a commercial sequencing facility ( macrogen , inc . , serum samples were also tested for zika virus igm , dengue virus ( denv ) , and japanese encephalitis virus ( jev ) by using an in - house igm capture elisa ( mac - elisa ) ( 13 ) . the interval between the date of symptom onset and the date of sampling ranged from 0 to 26 days ( n = 1,922 ; mean 4.13 days , 95% ci 1.666.6 days ) . five human serum samples were positive for zika virus by qrt - pcr ; these samples were collected in 2007 ( n = 1 ) , 2008 ( n = 1 ) , 2009 ( n = 2 ) , and 2015 ( n = 1 ) ( technical appendix 2 ) . no other samples , including urine and mosquito samples , were positive . among the 5 positive samples , phylogenetic analysis of partial sequences of the nonstructural protein 5 gene from human samples ( 1 from 2008 and 2 from 2009 ) showed that all 3 strains belonged to the asia genotype . kx455424 ) isolated in 2008 clustered closest with strains from the outbreak in micronesia ( 2007 ) ; 1 of the strains isolated in 2009 , t0706225 ( accession no . kx455425 ) , clustered closest with outbreak or endemic strains from new caledonia ( 2014 ) , thailand ( 2014 ) , china ( 2016 ) , chile ( 2014 ) , and french polynesia ( 2013 ) ; and the other strain isolated from 2009 , t1002464 ( accession no . kx455426 ) , clustered closest with the cambodia strain previously isolated in 2010 ( 9 ) ( figure 1 ) . these findings suggests that zika virus strains in cambodia are closely related to strains causing outbreaks in the pacific and south america and to endemic strains circulating in asia ; however , longer sequences are needed to confirm this finding . phylogenetic tree of zika virus partial nonstructural protein 5 gene of 3 strains detected from humans in cambodia . the partial sequences of the nonstructural protein 5 gene ( 192194 nt ) generated from the pcr products obtained for each strain were analyzed and assembled by using clc main workbench 5.5 package ( clc bio a / s , aarhus , denmark ) . mega6 ( 14 ) was used to perform multiple sequence alignment of cambodia strains with zika virus reference strains from africa and asia genotypes available in genbank and phylogenetic analyses by using maximum - likelihood method using the general time reversible model with 1,000 bootstrap resampling . cambodia strains from this study are indicated by bold type and a black triangle , and the cambodia strain isolated in 2010 is indicated by bold type and a white triangle . the serologic study of 1,992 acute - phase serum samples showed that 16 samples from 2007 ( n = 2 ) , 2008 ( n = 1 ) , 2010 ( n = 2 ) , 2012 ( n = 4 ) , and 2015 ( n = 7 ) were positive for zika virus igm , with no cross - reactivity with denv and jev , suggesting a presumptive diagnosis of recent infection . the interpretation of the 1,976 remaining samples is distributed as follows : 146 recent denv infections ; 4 recent jev infections ; 743 recent flavivirus infections ( defined by igm detection for > 2 of 3 antigens tested [ denv , jev , and zika virus ] ) ; and 1,087 samples negative for antibodies for any of the 3 viruses tested . the pcr- and igm - positive zika virus infections we detected came from 9 provinces in north , central , and south cambodia ( figure 2 ) . all 5 acute - phase samples that tested positive by rt - pcr were negative by mac - elisa . the convalescent - phase serum samples exhibited a pattern of recent flavivirus infection in 2 samples and recent zika virus infection in 1 sample ( technical appendix 2 ) . the confirmed case in 2015 was considered to be an autochthonous case because no history of recent travel to zika - endemic areas was reported by the patient . during january  may 2016 , all suspected dengue samples that tested negative for denv were also tested for zika virus ; no cases were detected , either by serology or rt - pcr . pcr- and igm - positive cases were from 9 different provinces in north , central and south cambodia . the 5 zika virus  positive samples by quantitative real - time reverse transcription ( qrt - pcr ) in this study were distributed as follows : the 2007 ( n = 1 ) sample was received from kampong cham province , and the other cases from 2008 ( n = 1 ) , 2009 ( n = 2 ) , and 2015 ( n = 1 ) were from the phnom penh area ( red star ) . the first case of zika virus infection previously reported in cambodia by conventional rt - pcr was diagnosed in a patient from kampong speu province ( 9 ) . the 16 additional serum samples found to be positive for zika virus igm were , from oldest to the most recent , from phnom penh ( 2007 , n = 1 ; 2008 , n = 1 ; 2010 , n = 2 ) ; battambang ( 2007 , n = 1 ; 2012 , n = 1 ) ; takeo ( 2012 , n = 1 ) ; kampong speu ( 2012 , n = 1 ) ; kampot ( 2012 , n = 1 ; 2015 , n = 1 ) ; kampong chhnang ( 2015 , n = 1 ) ; banteay meanchey ( 2015 , n = 1 ) ; and siem reap ( 2015 , n = 4 ) provinces . light gray indicates provinces with igm - positive cases , dark gray indicates province with pcr - positive cases , and black indicates provinces with pcr - positive and igm - positive cases . we show that zika virus circulated across cambodia during the past 10 years with low intensity and limited effect on public health . no clear explanation exists for the low detection of zika virus by rt - pcr or by mac - elisa , but it might be attributable to 1 ) the low incidence of zika virus , which usually causes mild disease compared with denv , in cambodia ; 2 ) possible bias in the selection of viremic cases , which were mainly reported in hospitalized suspected dengue patients and therefore were more severe ; 3 ) less than ideal timing of specimen collection for the detection of zika virus , which has been shown to be transient and frequently low - titer and too early for detection of zika virus igm on acute - phase serum samples ; or 4 ) the difficulty in interpreting mac - elisa results owing to the cross - reactivity of igm between denv and jev , which are widely present in cambodia . the epidemiology of zika virus is changing , and the risk for reintroduction in cambodia is a real threat that should not be underestimated . thailand , vietnam , and laos have reported cases since december 2015 , and , local transmission has been observed recently in singapore ( 5,15 ) . the risk for reemergence or reintroduction of zika virus should be monitored by extending the existing surveillance program to ambulatory suspected dengue patients and should be completed by a focal entomological study . moreover , in the absence of a previously detected zika outbreak in cambodia , seroprevalence studies in the general population should help estimate the size of the population remaining susceptible to zika infection . samples collected from 5 persons in cambodia in 2007 , 2008 , 2009 , and 2015 that tested positive for zika virus by quantitative rt - pcr .
we describe a retrospective study on circulation of zika virus in cambodia during 20072016 among patients with dengue - like symptoms and aedes aegypti mosquitoes . 
 our findings suggest that zika virus in cambodia belongs to the asia genotype , is endemic , has low prevalence , and has had low - level impact on public health .