J Vet Sci.  2012 Jun;13(2):111-118. 10.4142/jvs.2012.13.2.111.

Isolation and genetic characterization of Japanese encephalitis virus from equines in India

• Gulati BR
• Singha H
• Singh BK
• Virmani N
• Kumar S
• Singh RK

• KMID: 1376184
• DOI: http://doi.org/10.4142/jvs.2012.13.2.111

Abstract

Japanese encephalitis (JE) is an important vector-borne viral disease of humans and horses in Asia. JE outbreaks occur regularly amongst humans in certain parts of India and sporadic cases occur among horses. In this study, JE seroprevalence and evidence of JE virus (JEV) infection among horses in Haryana (India) is described. Antibodies against JEV were detected in 67 out of 637 (10.5%) horses screened between 2006 and 2010. Two foals exhibiting neurological signs were positive for JEV RNA by RT-PCR; JEV was isolated from the serum of one of the foals collected on the second day of illness. This is the first report of JEV isolation from a horse in India. Furthermore, a pool of mosquitoes collected from the premises housing these foals was positive for JEV RNA by RT-PCR. Three structural genes, capsid (C), premembrane (prM), and envelope (E) of the isolated virus (JE/eq/India/H225/2009) spanning 2,500 nucleotides (from 134 to 2,633) were cloned and sequenced. BLAST results showed that these genes had a greater than 97% nucleotide sequence identity with different human JEV isolates from India. Phylogenetic analysis based on E- and C/prM genes indicated that the equine JEV isolate belonged to genotype III and was closely related to the Vellore group of JEV isolates from India.

Keyword

envelope gene; horse; isolation; Japanese encephalitis virus; phylogenetic analysis

MeSH Terms

Animals
Antibodies, Monoclonal
Cloning, Molecular
Culex/virology
Encephalitis Virus, Japanese/*genetics/*isolation & purification
Encephalitis, Japanese/epidemiology/*veterinary/virology
Enzyme-Linked Immunosorbent Assay/methods/veterinary
Female
Genes, Viral
Genotype
Horse Diseases/epidemiology/*virology
Horses
India/epidemiology
RNA, Viral/genetics/isolation & purification
Reverse Transcriptase Polymerase Chain Reaction/veterinary
Seroepidemiologic Studies

Figure

• 

  Fig. 1 RT-PCR for detecting Flavivirus 3'NTR viral RNA (A) and Japanese encephalitis virus (JEV) E gene RNA (B). lane 1: serum from foal H225 on day 3 of illness, lane 2: brain homogenate from mice inoculated with H225 serum, lane 3: normal negative equine serum, lane 4: lysates from PS cells infected with JEV/eq/India/H225/2009 at passage 4, lane 5: serum from foal H238 on day 2 of illness, lane 6: uninfected PS cells, lane 7: JEVP20778 strain (reference positive control).

• 

  Fig. 2 Phylogenetic relationships of JEV/eq/India/H225/2009 with other JEV strains based on complete E (A) and prM (B) gene sequences. A maximum parsimony tree was generated using ClustalW. The tree was rooted using the Murray Valley encephalitis virus (strain MVE-1-51) sequence. Bootstrap confidence limits for 1,000 replicates are indicated above each branch. Each strain is noted with the species source, followed by country, isolate number, and year of isolation. GI~GIV: genotype I~IV.

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