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Am. J. Trop. Med. Hyg., 77(6), 2007, pp. 1139-1145
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

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Detection and Isolation of Japanese Encephalitis Virus From Blood Clots Collected During the Acute Phase of Infection

Gajanan N. Sapkal, Nitin S. Wairagkar, Vijay M. Ayachit, Vijay P. Bondre, AND Milind M. Gore*
National Institute of Virology, Pune, Maharashtra, India

Clinical specimens from an encephalitis outbreak in the Lakhimpur area of Uttar Pradesh, India, were investigated for identification and characterization of the etiologic agent. IgM capture ELISA showed recent Japanese encephalitis virus (JEV) infection. JEV isolation was attempted from white blood cells (WBCs) separated from blood clots of 12 patients (9 IgM positive and 3 negative) by serial co-culturing with phytohemagglutinin P–stimulated peripheral blood mononuclear leukocytes (PBMCs) obtained from pre-screened JEV sero-negative healthy individuals. JEV was isolated from two IgM-positive blood clots. Isolate 014178 was detected in WBCs and in the first passage of PBMCs by ELISA and reverse transcriptase-polymerase chain reaction. Isolate 014173 was detectable only after a second passage in PBMC co-culture. Sequence analysis of 346 nt of the C-prM region showed homology with JEV strain GP78. This is the first report on isolation of JEV from patient blood clots. Our study shows that the co-cultures of PBMCs separated from patient blood clots provide an additional source for JEV isolation.

Received May 11, 2007. Accepted for publication August 24, 2007.

Acknowledgments: We thank Pradip Fulmali, J. P. Thakare, S. D. Pawar, and S. A. Mahamuni for invaluable technical support. The valuable suggestions by A. C. Mishra, Director, National Institute of Virology, Pune, are greatly appreciated.

* Address correspondence to Milind M. Gore, National Institute of Virology, Indian Council of Medical Research, 20-A, Dr. Ambedkar Road, P.B.No.11, Pune 411001, India. E-mail: milind_gore{at}hotmail.com

Authors’ addresses: Gajanan Sapkal, Nitin Wairagkar, Vijay Ayachit, Vijay Bondre, and Milind Gore, National Institute of Virology, Sus Road, Pashan, Pune, Maharashtra 411021 India, Telephone: +91-20-25871982, Fax: +91-20-25871895, E-mail: milind_gore{at}hotmail.com.




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V. P. Bondre, G. N. Sapkal, P. N. Yergolkar, P. V. Fulmali, V. Sankararaman, V. M. Ayachit, A. C. Mishra, and M. M. Gore
Genetic characterization of Bagaza virus (BAGV) isolated in India and evidence of anti-BAGV antibodies in sera collected from encephalitis patients
J. Gen. Virol., November 1, 2009; 90(11): 2644 - 2649.
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Copyright © 2007 by the American Society of Tropical Medicine and Hygiene.