Genetic Variants of Echovirus 13, Northern India, 2010

Nonpolio acute flaccid paralysis is increasing in India. To determine viral causes, we conducted cell culture and molecular analysis identification of nonpolio human enteroviruses associated with acute flaccid paralysis during March–August 2010 in northern India. The predominant nonpolio enterovirus found was echovirus 13, a serotype rarely isolated in India.

Nonpolio acute flaccid paralysis is increasing in India.
To determine viral causes, we conducted cell culture and molecular analysis identification of nonpolio human enteroviruses associated with acute flaccid paralysis during March-August 2010 in northern India. The predominant nonpolio enterovirus found was echovirus 13, a serotype rarely isolated in India.
A lthough polio has not been reported from India since January 2011, spurts in the rate of nonpolio acute flaccid paralysis (AFP) are of concern. Therefore, other viral agents associated with AFP need to be identified, especially nonpolio human enteroviruses (HEVs), which are a major cause of neurologic illness.
The classic method for HEV serotypic identification requires virus isolation in susceptible cell cultures, followed by virus neutralization tests. However, the procedure is laborious, slow, and incapable of identifying new serotypes for which no reference antiserum is available (1). Nonpolio HEV typing based on viral protein (VP) 1 sequences has been developed and correlates well with antigenically defined serotypes (2).
Since 1998, we have been actively involved in polio surveillance and passively reporting nonpolio HEV activity in Uttar Pradesh State, northern India. Most nonpolio HEVs were isolated during the summer-autumn season and identified by using methods based on antigenic serotyping (3). We used a method combining a single cell culture passage with VP1 reverse transcription PCR (RT-PCR) and sequencing for rapid identification of nonpolio HEV serotypes. The predominant nonpolio HEV found was echovirus (E) 13, a serotype rarely isolated in India.

The Study
Nonpolio AFP case-patients showing signs of acute onset of focal weakness or paralysis characterized as flaccid (reduced muscle tone) and preceded by fever were considered for NPEV analysis. A total of 347 fecal specimens collected in Uttar Pradesh during March-August 2010 from children <15 years of age who had AFP symptoms were analyzed by using a modified World Health Organization (WHO) method (4,5).
Fecal specimens were processed according to the WHO protocol (4). Human rhabdomyosarcoma and L20B (mouse fibroblast cells expressing the poliovirus receptor) cell lines used for virus isolation were observed for cytopathic effect (CPE). The positive isolates with enterovirus (EV)-like CPE were tested by using pan-poliovirus and pan-EV RT-PCR (4). All poliovirus isolates characterized according to WHO algorithm were excluded from the analysis presented here. Viral RNA was extracted from the firstpass rhabdomyosarcoma cell suspension with the Viral RNA Mini Kit (QIAamp, QIAGEN, Hilden, Germany). Genotyping was done by RT-PCR of a portion of the VP1 gene and sequencing as described (2,6).
MEGA version 5.05 software (7) was used for phylogenetic analysis. Distance matrices of the nucleotide and amino acid sequences were analyzed with the MegAlign program in the Lasergene software package (DNASTAR, Madison, WI, USA).
The 244-nt partial VP1 gene sequence of all the Uttar Pradesh E13 isolates shared 75.4%-79.9% nt identity and 87.7%-93.8% aa identity with the prototype EV13 strain, Del Carmen (GenBank accession no. AY302539). Phylogenetic analysis indicated the existence of 2 different Uttar Pradesh E13 clades in the neighborjoining tree (Figure). Uttar Pradesh 2010 E13 viruses in the largest clade segregated into 3 groups (labeled A-C, Figure), whereas the second large 2010 Uttar Pradesh E13 clade was monophyletic (labeled D, Figure).

Conclusions
We demonstrated the utility of a modified WHO laboratory protocol by combining classical and molecular methods for rapid detection and identification of nonpolio HEV (2,(4)(5)(6). Identifying the EV in mixed EV infections is challenging when this protocol is used, but on balance, pan-EV testing followed by VP1 RT-PCR and sequencing delivers rapid results and more efficient use of labor and resources than does the traditional method and enables identification of newer EV types.
Numerous  EVs (3,8). Nonpolio HEVs also have been detected in epidemics of paralytic disease (9). Although nonpolio HEVs can be isolated from asymptomatic children, few studies have compared the nonpolio HEV serotypes found in AFP case-patients and in healthy children. Identifying nonpolio HEV serotypes associated with AFP in India provides useful EV surveillance data and should be explored further in conjunction with virologic evaluation of appropriate community controls and additional clinical specimens from AFP case-patients (cerebrospinal fluid, throat swab samples, serum), which could indicate the nonpolio HEV type causing paralytic disease. We identified 8 nonpolio HEV serotypes (E4, E7, E13, E25, E33, EV75, CVB3, and CVB6). E13, the predominant serotype, has been isolated rarely (3,10); however, nonpolio HEVs have been reported from India in connection with sporadic and epidemic meningitis/encephalitis cases (11,12). A wide spectrum of illnesses have been reported with E13 (13), so finding E13-associated AFP cases mimicking poliomyelitis (14) is not surprising.
The global aseptic meningitis epidemic caused by E13 during 2000-2003 was associated with a single E13 genotype (15); however, our findings indicate that several different and genetically diverse E13s have been cocirculating in India. The Uttar Pradesh E13 partial VP1 gene sequences are most closely related to recent E13 strains from central and southern Asia. The high genetic diversity among the Uttar Pradesh E13 isolated from patients in a local area over 6 months implies a continuous pattern of circulation. High-density human populations, immunologically susceptible cohorts, and nonhygienic environmental conditions probably facilitated the E13 genetic heterogeneity in our study.
Although we report a high prevalence of E13 infection in AFP case-patients, our study is restricted by the modest number of cases from a small geographic area sampled over a short period. The epidemiologic and nonpolio HEV genetic information gathered warrants further studies in larger groups of children to gain better insight into AFP caused by nonpolio HEV and to identify the etiologic nonpolio HEV type(s) associated with sporadic or epidemic AFP as India nears the goal of polio elimination.  Figure. Phylogenetic tree based on alignments of partial viral protein 1 gene sequences of echovirus 13 (E13) constructed by the neighbor-joining method implemented in MEGA version 5.05 software (7) by using the Kimura-2 parameter nucleotide substitution model. Bootstrap analysis included 1,000 pseudoreplicate datasets. Clusters are labeled A, B, C, and D. Square indicates Uttar Pradesh E13 from fecal samples. All Uttar Pradesh E13 isolates on the tree are identified by using the same numbers listed in Table 1. Triangle indicates E13 prototype Del Carmen and the out-group enterovirus 69. Scale bar indicates nucleotide substitutions per site.