a total of 850 specimens collected from 306 patients who had encephalitis included 306 csf specimens , 304 blood samples , 120 throat swabs , and 120 rectal swabs .
all samples were stored at 20c before being transported for analysis and thereafter were stored at 70c at the national institute of virology in pune , india .
laboratory tests conducted by state government health services of uttar pradesh were negative for bacteria and malaria . according to standard protocol ( 2 ) ,
virus isolation was attempted in human rhabdosarcoma ( rd ) and in baby hamster kidney ( bhk ) cell lines .
separate aliquots were processed in 2 laboratories to maintain quality control and monitor possible contamination during pcr processing .
viral nucleic acids were extracted by using viral rna mini kits ( qiaamp , qiagen , hilden , germany ) .
rt - pcr was performed for ev by using 5 noncoding region ( ncr)specific primers , as has been described ( 5,6 ) .
genotyping was conducted by using rt - pcr of virion protein ( vp ) 1/2a and vp1 regions and sequencing ( 7,8 ) .
table 1 describes the locations and sequences of the primers used in the assays . *
ncr , noncoding region of viral genome ; vp , virion protein ; na , not applicable .
pcr products were purified by using a gel extraction kit ( qiaquick , qiagen ) .
both strands were sequenced by using bigdye terminator cycle sequencing ready reaction kit ( applied biosystems , carlsbad , ca , usa ) in abi prism 3130 xl genetic analyser ( applied biosystems ) .
mega 3.1 software generated the phylogenetic tree by using the neighbor - joining algorithm and kimura 2parameter distance model and applying a bootstrap test that used 1,000 bootstrap replications ( 9 ) .
clinical histories available for 253 of the 306 patients showed fever and altered sensorium in 100.0% , hepatomegaly in 70 ( 27.8% ) , splenomegaly in 49 ( 19.4% ) , and meningeal signs in 35 ( 13.9% ) of the 253 patients .
specimens that were adequate for isolation included 85 of 306 csf specimens , 18 of 304 serum samples , 19 of 120 rectal swabs , and 19 of 120 throat swabs .
cytopathic effect was observed in cell cultures inoculated with 4 csf specimens , 2 rectal swabs , 2 throat swabs , and 1 serum sample .
electron microscopic examination of cultures infected with 2 csf samples showed picornavirus - like particles 2527 nm in diameter .
attempts to detect ev rna in the isolates and clinical specimens used nested rt - pcr in 5 ncr .
sequences of amplicons from 3 csf specimens and 2 rectal swabs showed 97.2%98.9% homology with ev-89 ( i.e. , the strain named banoo-10359 , genbank accession no .
ay697459 ) and 95.7%96.9% homology with ev-76 ( fra91 - 10369 , genbank accession no .
sequences from 1 isolate from a csf specimen and 1 isolate from a rectal swab showed 100.0% homology with coxsackie virus b3 ( cv - b3 ) strain 20 .
one isolate from serum showed 98.3% homology with coxsackie virus b1 ( cv - b1 ) strain samp2.17 .
sixty - six ( 21.5% ) of 306 csf specimens , 7 ( 6.4% ) of 110 rectal swabs , 4 ( 3.7% ) of 110 throat swabs , and 1 ( 5.5% ) of 18 serum samples showed amplification in 5 ncr of the ev genome .
sequences of 64 of 78 ( 82.0% ) pcr products ( 59 from csf specimens , 4 from rectal swabs , and 1 from a throat swab ) showed 97.2%98.9% and 95.7%96.9% homology with ev-89 and ev-76 , respectively . ten ( 12.8% ) products ( 7 from csf , 2 from rectal swabs , and 1 from serum ) showed 99.3%100.0% homology with cv - b3 ( figure 1 ) .
three pcr products , each derived from a throat swab , showed 93.3%96.6% homology with coxsackie virus a ( cv - a ) , echovirus 11 , and echovirus 30 , respectively .
phylogenetic tree based on partial 5 noncoding region sequences of enterovirus ( ev ) genome detected in cerebrospinal fluid samples from encephalitis patients .
specimens are identified by repository serial numbers obtained from the national institute of virology ( niv ) , pune , india .
ev , enterovirus ; csf , cerebrospinal fluid ; cv - a , coxsackie virus a ; cv - b , coxsackie virus b ; hev , human enterovirus .
isolates from 2 of 5 cell cultures , 2 of 59 csf specimens , and 1 of 4 rectal swabs contained ev-76 .
two of 4 rectal swabs were characterized as ev-89 on the basis of partial vp1/2a ( 29173374 ) or vp1 ( 26022977 ) gene sequences .
ay697463 , ay697464 , ay697471 , ay697469 , ay697462 , and ay697468 ) and 93.6%94.5% homology with ev-89 strain ( genbank accession no .
ay697459 ) ( figure 2 ) . within ev-76 and ev-89 strains of the study ,
attempts to amplify vp1/2a or vp1 regions of ev rna detected in most clinical specimens failed despite the use of sensitive primer pairs that have been discussed recently ( 10 ) .
phylogenetic tree based on partial virion protein 1 ( vp1 ) sequences ( 26022977 ) detected in enterovirus ( ev ) isolates and clinical specimens from encephalitis patients .
ev , enterovirus ; cv - a , coxsackie virus a ; cv - b , coxsackie virus b ; hev , human enterovirus ; niv , national institute of virology , pune , india .
table 2 describes details of clinical findings in the subsets of ev - positive and ev - negative specimens of the patients for whom clinical histories were available .
further , hepatomegaly and splenomegaly appeared to be proportionately higher in patients with enteroviral infections than in patients whose specimens were negative for ev and je virus .
the viral rna detected in csf samples from patients hospitalized with encephalitis in uttar pradesh showed close identity with the ev-89 and ev-76 that recently were reported as an unusual group classified genetically as group a ev ( ev - a ) ( 10 ) . presence of the virus was also confirmed by its isolation and typing .
human ev-76 was detected in isolates in 1 rectal swab and 2 csf specimens , and human ev-89 was detected in 2 rectal swabs by using amplification of vp1/2a or vp1 regions .
sequence analysis showed nt homology of 92.7%97.7% with bangladesh ev-76 and ev-89 strains recovered from patients with acute flaccid paralysis ( afp ) .
the failure of amplification of typing regions in most specimens may be due to a low viral load .
evs are known to cause severe neurologic diseases ranging from afp to encephalitis ( 11 ) . in recent years
, southeast asian countries have reported outbreaks of encephalitis caused by ev-71 ( 12,13 ) . during afp surveillance activities ,
afp patients infected with echoviruses and coxsackie b viruses also have been detected in india ( 15 ) .
isolation of ev from clinical specimens collected from children with encephalitis in the present study indicates viable virus .
detection of ev-89/76 rna in the csf of 20% of the patients suggests the association of these viruses with encephalitis . also , in 10 ( 3.3% ) of 306 patients , co - infections of je virus and ev were detected .
further studies are needed to understand the relative contributions of these viruses in causing sporadic and outbreak infections of encephalitis .
accumulation of water in a saucer - shaped landscape ( terai ) and extensive rice cultivation in eastern uttar pradesh and adjoining regions favor the growth of vector mosquito populations and waterborne pathogens . though the source of infection in the present study is unclear , the data warrant active surveillance of encephalitis cases .
inadequate hygiene and the unsanitary conditions that prevail in the study region may encourage the spread of ev infections in the community .
studies conducted on environmental samples may provide clues related to the dynamics of ev infections in humans .
 an outbreak of viral encephalitis occurred in northern india in 2006 . attempts to identify an etiologic agent in cerebrospinal fluid by using reverse transcription  
 pcr showed positivity to enterovirus ( ev ) in 66 ( 21.6% ) of 306 patients . 
 sequencing and phylogenetic analyses of pcr products from 59 ( 89.3% ) of 66 specimens showed similarity with ev-89 and ev-76 sequences . 
The Study
Conclusions