Japanese encephalitis virus is the most important cause of epidemic encephalitis worldwide with an estimated 30,000–50,000 cases annually . The geographical area affected by the virus is expanding, and despite the availability of vaccines, JE is a growing public health problem [17, 18]. West Nile virus, until recently being a relatively benign virus, causing epidemics of a fever-arthralgia-rash syndrome, and only occasional CNS disease  has disproved this belief with the recent outbreaks of WN encephalitis in Romania and New York [20, 21]. These viral encephalitic conditions are acute in onset and may mimic other acute infectious conditions of CNS such as, Tubercular meningitis, cerebral malaria and other viral encephalitis . The need for an accurate and rapid diagnostic test is much sought for.
Conceptually, the most rapid diagnosis of an arbovirus infection can be made by direct detection of virus antigen or nucleic acid in clinical specimens. Rapid serologic diagnostic tests, such as Enzyme Immuno-Assay (EIA) can provide strong presumptive etiologic evidence, if specific IgM is detected in the acute-phase serum or CSF specimen. Detection of IgM is not always evidence of current infection with certain arboviruses, especially flaviviruses, which can induce persistent IgM production [23, 8]. Genome detection, though very useful in diagnosing WN encephalitis, is of limited value in diagnosing JE and Dengue encephalitis .
Virus isolation, though more specific compared to antigen detection, is less sensitive and time consuming requiring minimum of 3–7 days compared to 5–6 hours required for antigen detection by indirect immuno- fluorescent technique . Shell vial culture, a modification of conventional cell culture technique works on the principle that centrifugation mechanical force enhances the viral infectivity to the susceptible cells . This technique has been used for the rapid diagnosis of infections by various viruses such as cytomegalovirus, Herpes Zoster, Mumps, Measles and respiratory syncytial virus . In all these studies, shell vial culture technique has been shown to increase the rate of isolation of the viruses without any compromise on the specificity. Shell vial culture technique also has been shown to significantly reduce the time taken as compared to conventional cell culture technique.
In the present study that included 50 patients, males were 32 (64%), and females were 18(36%) with a male to female ratio of 1.7:1. Pediatric age group (0–12 years) was the predominant one compared to the adults. This predominant involvement of pediatric age group (92%) and the Male:Female ratio of 1.7:1 are in accordance with the age and sex distribution of the earlier epidemics of viral encephalitis in the endemic areas including Pondicherry and Tamilnadu . Viral etiology could be proved in 29(58%) out of 50 cases, of which 28 (96.5%) belonged to the pediatric age group. JEV was the commonest etiological agent (38%) followed by WNV (18%) and Den-2 virus in one case. These findings suggest a high incidence of JE and WN encephalitis in this region of South India. Endemicity of JEV in South India is a known fact. There have been earlier documented cases of WN encephalitis from South India [26, 27]. A recent report from India  documents 88 sporadic cases of WN infection including 7 cases of WN encephalitis. This suggests that WNV is active and prevalent in India. Though Dengue infection is present since ancient times in India, documented encephalitic form is rare. Isolated reports of Dengue encephalitis from India  suggest that it might be an under -diagnosed clinical entity, because of lack of proper laboratory facilities.
A total of 9 (65.5%) out of 19 patients positive for Japanese encephalitis virus had neurological deficits. Focal neurological deficits occurred in 6(31.5%) patients who recovered from Japanese encephalitis. The neurological deficits that were noted in the study group were facial palsy (n = 6), hemiparesis (n = 3) and hemiplegia (n = 1). All the neurological deficits were found in patients positive for Japanese encephalitis virus. In one case facial palsy was associated with hemiplegia. No neurological deficits were noticed in patients positive for West Nile or Den-2 virus infections. It is interesting to note the absence of neurological deficits like flaccid paralysis in WN encephalitis cases that is described in outbreaks of WN virus infection in the United States[30, 31].
Among the 50 patients included in the study, 4 (8%) patients left against medical advice, so the clinical outcome of these cases is not known. Out of the remaining 46 (92%), 16(34.8%) expired and the rest 30(65.2%) had a clinical recovery with or without neurological sequelae. 8 patients positive for Japanese encephalitis virus expired with a case fatality rate (CFR) of 50%. In the present study, West Nile encephalitis had a CFR of 62.5%. Dengue-2 infection was detected only in one patient, who showed complete clinical recovery. This high case fatality rate among children with West Nile encephalitis is in contrast to other published reports where in advanced age is the most important risk factor for morbidity and mortality. It is unclear if this high mortality rate among the children is due to a highly virulent strain of the virus.
Indirect immunofluorescence test, the best-studied antigen detection method for the diagnosis of viral encephalitis by flaviviruses especially Japanese encephalitis , could detect the viral antigens in 24(48%) cases. This finding correlates with the earlier IIF studies for the diagnosis of viral encephalitis [32, 33].
Shell Vial Culture could demonstrate viral etiology in all the 24 cases positive by IIF. In addition shell vial culture could detect the virus in 5(10%) more cases, which IIF failed to detect. Therefore, the technique could establish viral etiology in 29(52%) cases compared to 24(48%) by IIF. The significant reduction in the time taken (36 hours), compared to conventional cell culture technique (3–7 days) is an important advantage, which could be due to the hastening of viral entry into the cells of monolayer, as hypothesized. In order to ensure a rapid and precise diagnosis, early, prompt collection, transport and processing of the CSF samples becomes mandatory.