Detection & differentiation of Coxsackie A 24 variant isolated from an epidemic of acute haemorrhagic conjunctivitis in north India by RT-PCR using a novel primer pair.
OBJECTIVE
An epidemic of acute haemorrhagic conjunctivitis (AHC) occurred in north India during July to September 1994. We report a reverse transcription-polymerase chain reaction (RT-PCR) using known and novel primers to differentiate and identify the CA 24 virus isolated from the epidemic of AHC.


METHODS
Conjunctival swabs were collected from 46 patients (in 12 patients from both the eyes) yielding 58 swabs. The swabs were inoculated in RD 19S and HeLa-199 cell monolayers and observed for cytopathic effect. Serum neutralizing antibodies were tested in 17 acute and 10 convalescent phase serum samples. RT-PCR was done on 9 isolates (7 Coxsackie A 24 and 2 ECHO-1 as identified by neutralization test) using known and a novel primer. Fourteen virus isolates (9 CA 24, 3 ECHO-1 and 2 untyped) were inoculated in suckling mice and these mice were observed daily for 10 days for flaccid paralysis of hind limb or death.


RESULTS
Cytopathic virus was isolated from conjunctival swabs in 21 of 46 (45.6%) patients subjected to virus isolation. Sixteen of 21 (76.2%) isolates were neutralized by CA 24 specific antisera, 3 isolates were identified as ECHO-1 with Schmidt enteroviruses antiserum pools while 2 remained untypable. Of these 21 isolates, 9 representative isolates (7 CA 24 and 2 ECHO-1) tested by RT-PCR had enterovirus common region DNA but did not show any amplification in RT-PCR with EV-70 specific primers (VP-1 and VP-3). Using CA 24 specific novel (VP 3-1) primers amplification was seen in 6 of 7 CA 24 isolates while 2 ECHO-1 remained unamplified. In contrast with 3C-proteinase region primers, only 2 of 7 CA 24 were amplified along with false amplification of both ECHO-1. Serum neutralizing antibodies were seen in 2 of 17 (11.7%) acute phase sera and 6 of the 10 (60%) convalescent phase sera while in paired sera (available in two patients) a four-fold rise in titres were observed. Hind-limb paralysis and/or death occurred in all suckling mice inoculated with CA 24 isolates while mice remained healthy after inoculation with 3 isolates of ECHO-1 and 2 untypable isolates.


CONCLUSIONS
The epidemic of AHC was caused by a variant of CA 24. Molecular typing can detect and differentiate between CA 24 and EV-70 viruses. Novel primer pair was found useful in the identification and confirmation of CA 24 isolates.
D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D003232:Conjunctivitis, Acute Hemorrhagic / Q000453:epidemiology* / Q000821:virology; D003384:Coxsackievirus Infections / Q000453:epidemiology* / Q000821:virology; D017931:DNA Primers; D004196:Disease Outbreaks; D030015:Enterovirus C, Human / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D006801:Humans; D007194:India / Q000453:epidemiology; D008297:Male; D051379:Mice; D008875:Middle Aged; D009500:Neutralization Tests; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers