at least 442 denv-1 isolates from the 20042007 dengue outbreak were obtained from the diagnostic virology repository at the university of malaya medical centre .
viral rna was extracted from infected cell culture supernatants , and a 1-step reverse transcription 
pcr amplification of the denv-1 envelope gene was performed by using amplification primers ( 8) .
genome sequences from study isolates and those obtained from genbank ( table 1 ) were used to construct phylogenetic trees .
maximum clade credibility was inferred by using the bayesian markov chain monte carlo method implemented in beast version 1.5.2 ( 9 ) . for simplicity , only 10 new denv-1 sequences from the study and 47 from genbank were analyzed .
phylogenetic trees showed 6 distinct denv-1 subgenotypes : 3 ancestral subgenotypes ( hawaii / japan , 1940s ; thailand , 1960s ; and malaysia , 1972 ) and 3 major endemic subgenotypes ( si , sii , and siii ) , which is consistent with reported findings ( 8) . an isolate identified as d1.malaysia.36046/05 grouped with isolate p72_1244 , a sylvatic denv-1 reportedly isolated from a sentinel monkey in malaysia in 1972 .
virus envelope gene sequence shared > 97% nt sequence similarities and > 99% aa sequence similarities .
there was only 1 aa difference at position 55 , from valine in p72_1244 to isoleucine in d1.malaysia.36046/05 .
focus - reduction neutralization tests ( frnts ) were performed by using the d1.malaysia.36046/05 isolate .
serum samples from patients with primary dengue caused by denv-1 si and sii ( figure ) were pooled and used in frnts as described ( 10 ) . neutralizing antibody titer
was defined as the reciprocal of the highest serum dilution that reduced viral foci by 50% ( frnt50 ) .
frnt results after adjustment of the titer to that of respective isolates showed that the d1.malaysia.36046/05 virus is neutralized by serum from patients with denv-1 si infections ( frnt50 = 320 ) and samples from patients with denv-1 sii infections ( frnt50 = 80 ) ( table 2 ) .
maximum clade credibility tree of complete envelope genes of dengue virus type 1 ( denv-1 ) isolates .
coalescent times with 95% highest posterior density values ( ranges in parentheses ) and posterior probability values ( all 1.0 ) of key nodes are shown .
patient convalescent - phase serum samples used for neutralization assays from which virus was isolated are indicated at the end of branches according to their virus groups .
sii , subgenotype ii , si , subgenotype i. * mock , controls treated with serum from healthy ( no dengue infection ) donors ; virus , virus plus diluent ; medium , serum and diluent without virus ; si , subgenotype i ; sii , subgenotype ii .  reciprocal of the highest serum dilution that reduced viral foci by 50% ( 50% focus - reduction neutralization test ) .
virus was treated with serum from patients infected with primary dengue virus type 1 si or sii .
laboratory and clinical records showed that d1.malaysia.36046/05 virus was isolated from a patient who had headache , body ache , chills , rigors , and abdominal pain for 3 days and sought treatment at the university of malaya medical centre .
the patient did not return for subsequent follow - up , and efforts to locate the patient were unsuccessful .
the most recent address of the patient was within a high population  density area of kuala lumpur .
additional sequencing of other denv-1 isolates from the 20042007 outbreak did not identify any additional d1.malaysia.36046/05like virus .
isolation of the ancestral denv-1 after > 30 years suggests that a mosquito  host transmission cycle has maintained this virus .
the natural host of the virus can not be determined conclusively because the only known fact is that the virus was isolated from a patient with dengue fever .
the original ancestral denv-1 isolate p72_1244 was designated as sylvatic because it was isolated from a sentinel monkey in a rural forest ( 3 ) .
its sylvatic origin has recently become uncertain because the virus genome is phylogenetically closer to other endemic denv-1 lineages ( 11 ) .
however , because no virus with high sequence similarities to that of denv-1 isolate p72_1244 has been isolated over the past 33 years , the virus may have been maintained in a sylvatic cycle through a nonhuman primate / mosquito enzootic cycle .
the estimated sequence evolution rate for d1.malaysia.36046/05 is 5.20  10 substitutions / site / year .
this rate is relatively slower than those for other endemic denv-1 isolates used in this study ( 5.67  10 to 8.05  10 substitutions / site / year ) .
the much smaller monkey : human population ratio ( 700,000:28,000,000 ) ( 12 ) ( http://en.wikipedia.org/wiki/malaysia ) and the more restricted mobility of monkeys could have limited the virus genome sequence divergence , leading to conservation of the sylvatic virus genome sequence .
the absence of the virus from the endemic urban cycle over the past 33 years could have been caused by its inability to overcome population herd immunity after exposure to endemic denv-1 .
efficient neutralization of virus by serum from patients infected with denv-1 si and sii supports this possibility ( 13 ) .
conversely , the virus may not be highly transmissible by peridomestic mosquitoes ( 14 ) and may be confined to the enzootic forest cycle .
therefore , isolation of the ancestral virus from a person living in kuala lumpur is most likely the result of a stochastic spillover event after contact with infected forest - dwelling mosquitoes .
available evidence does not support endemic presence of the virus in an urban dengue cycle .
however , a sylvatic cycle needs to be considered in any future dengue vaccination initiatives .
 ancestral sylvatic dengue virus type 1 , which was isolated from a monkey in 1972 , was isolated from a patient with dengue fever in malaysia . 
 the virus is neutralized by serum of patients with endemic denv-1 infection . 
 rare isolation of this virus suggests a limited spillover infection from an otherwise restricted sylvatic cycle . 
The Study
Conclusions