Viral screening
The lungs and spleens of aborted fetuses were immerged into precooled
phosphate-buffered saline (PBS, PH7.4) at 1:5 and grounded. After three
freeze-thaw cycles, the samples were centrifuged at 10,000 g for 10 min,
and the supernatant was stored at -70oC prior to RNA
extraction or virus isolation.
Total viral RNA was extracted from 300 ul of virus-containing
supernatant using Trizol reagent following manufacture instructions
(Invitrogen Carlsbad, CA). The reverse transcription reactions were
performed using random primers and M-MLV reverse transcriptase
(ThermoFisher Scientific China Ltd. Shanghai). CSFV was verified by the
specific primers reported previously (Yanting et al., 2009). GETV C gene
was amplified by forward primer ACCGAAGAAGCCGAAGAA and reverse primer
GCACTCRAGGTCATACTTG designed from the sequences of GETV HB0234
(accession number: EU015062) and 12IH26 (accession number: LC152056)
available in GenBank. The amplified products were analyzed by 1.5%
agarose gel electrophoresis and gel imaging (Sangon Biotech Co., Ltd.,
Shanghai).