Efficient rescue of foot-and-mouth disease virus in cultured cells transfected with RNA extracted from clinical samples.
In this study, an RNA transfection was used to rescue infectious foot-and-mouth disease (FMD) virus from clinical samples in BHK-21 cell line for diagnosis of FMD. Tissue samples (n=190) were subjected to FMD virus isolation by conventional cell culture and also by RNA transfection. FMD virus was isolated from 62% of the clinical samples by RNA transfection, whereas virus was isolated only from 16% of the clinical samples in conventional cell culture method, suggesting better performance of the RNA transfection. Virus was rescued from 67% and 10% of ELISA negative but multiplex PCR positive samples by RNA transfection and conventional cell culture, respectively. The efficiency of transfection was studied on clinical samples subjected to temperature as high as 37°C and varying pH (pH 4-9). Except up to 1 week of storage at 4°C at pH 7.5, virus isolation was not possible by cell culture. Virus was rescued by transfection from samples stored at 4°C for any of the applied pH up to 4 weeks, and when stored at 37°C virus could be rescued up to 4 weeks at pH 7.5 suggesting the fitness of transfection to isolate virus from clinical samples stored under inappropriate conditions. The sequence data and antigenic relationships with the vaccine strains, between virus rescued by transfection and conventional cell culture, were comparable. The RNA transfection will help to increase the efficiency of virus isolation, diagnosis and molecular epidemiological studies.
Project Directorate on Foot and Mouth Disease, Mukteswar, Nainital 263138, Uttarakhand, India.
Punam
P
Bisht
Project Directorate on Foot and Mouth Disease, Mukteswar, Nainital 263138, Uttarakhand, India.
Jajati K
JK
Mohapatra
Project Directorate on Foot and Mouth Disease, Mukteswar, Nainital 263138, Uttarakhand, India.
Saravanan
S
Subramaniam
Project Directorate on Foot and Mouth Disease, Mukteswar, Nainital 263138, Uttarakhand, India.
Biswajit
B
Das
Project Directorate on Foot and Mouth Disease, Mukteswar, Nainital 263138, Uttarakhand, India.
Veena
V
Pande
Project Directorate on Foot and Mouth Disease, Mukteswar, Nainital 263138, Uttarakhand, India.
Jitendra K
JK
Biswal
Project Directorate on Foot and Mouth Disease, Mukteswar, Nainital 263138, Uttarakhand, India.
Gaurav K
GK
Sharma
Project Directorate on Foot and Mouth Disease, Mukteswar, Nainital 263138, Uttarakhand, India.
Manoranjan
M
Rout
Project Directorate on Foot and Mouth Disease, Mukteswar, Nainital 263138, Uttarakhand, India.
Rajeev
R
Ranjan
Project Directorate on Foot and Mouth Disease, Mukteswar, Nainital 263138, Uttarakhand, India.
Bana B
BB
Dash
Project Directorate on Foot and Mouth Disease, Mukteswar, Nainital 263138, Uttarakhand, India.
Aniket
A
Sanyal
Project Directorate on Foot and Mouth Disease, Mukteswar, Nainital 263138, Uttarakhand, India. Electronic address: pattnaikb@gmail.com.
Bramhadev
B
Pattnaik
D000818:Animals; D002460:Cell Line; D003955:Diagnostic Tests, Routine / Q000379:methods*; D005536:Foot-and-Mouth Disease / Q000175:diagnosis* / Q000821:virology*; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D012367:RNA, Viral / Q000235:genetics*; D012680:Sensitivity and Specificity; D013696:Temperature; D014162:Transfection; D014773:Virology / Q000379:methods*
D012367:RNA, Viral
Chemical method; Foot-and-mouth disease; RNA transfection; Rescue of virus