Bovine parainfluenza virus type 5 (bPIV5) was isolated from cattle with downer cow syndrome in 2012, and included both respiratory and neurotropic pathogens from a variety of animals. In the current study, we conducted serosurveillance using sera obtained from seven Korean farms and optimized a reve...
Bovine parainfluenza virus type 5 (bPIV5) was isolated from cattle with downer cow syndrome in 2012, and included both respiratory and neurotropic pathogens from a variety of animals. In the current study, we conducted serosurveillance using sera obtained from seven Korean farms and optimized a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect bPIV5. The overall seropositive rate for Korean cattle was 21.4% (163/760). A farm located near the city of Milyang in Gyeoungnam province had a markedly elevated seropositive rate for bPIV5 compared to that of the other six farms. The regional seropositive rates were 4.2% (8/192) for Haman, 19.5% (18/55) for Hwasung, 73.9% (65/88) for Milyang, 26.0% (50/192) for Namwon, 1.0% (1/96) for Uljin, 13.5% (13/96) for Yeongju, and 32.7% (8/41) for Yongin. The sensitivity and specificity of three RT-PCR primer sets used to amplify the conserved fusion gene of bPIV5 were also evaluated. An RT-PCR assay using the bPIVFR3 primer set was 10-fold more sensitive than the assays using the two other primer sets and did not result in non-specific amplification. These results demonstrated that the bPIFR3 primer set can be used to detect bPIV5.
Bovine parainfluenza virus type 5 (bPIV5) was isolated from cattle with downer cow syndrome in 2012, and included both respiratory and neurotropic pathogens from a variety of animals. In the current study, we conducted serosurveillance using sera obtained from seven Korean farms and optimized a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect bPIV5. The overall seropositive rate for Korean cattle was 21.4% (163/760). A farm located near the city of Milyang in Gyeoungnam province had a markedly elevated seropositive rate for bPIV5 compared to that of the other six farms. The regional seropositive rates were 4.2% (8/192) for Haman, 19.5% (18/55) for Hwasung, 73.9% (65/88) for Milyang, 26.0% (50/192) for Namwon, 1.0% (1/96) for Uljin, 13.5% (13/96) for Yeongju, and 32.7% (8/41) for Yongin. The sensitivity and specificity of three RT-PCR primer sets used to amplify the conserved fusion gene of bPIV5 were also evaluated. An RT-PCR assay using the bPIVFR3 primer set was 10-fold more sensitive than the assays using the two other primer sets and did not result in non-specific amplification. These results demonstrated that the bPIFR3 primer set can be used to detect bPIV5.
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제안 방법
An RT-PCR assay for the detection of bPIV5 was carried out using three primer sets to optimize assay sensitivity. The bPIV5 culture supernatant (QIA-B1201; 107.
The HA test was performed by preparing a serial twofold dilution of bPIV5 (strain QIA-B1201) into 50 µL of PBS (pH 7.2) with 50 µL of 0.6% chicken erythrocytes.
The HA titer was expressed as the reciprocal of the highest dilution of bPIV5 showing complete hemagglutination. The HI test was performed in 96-well microplates according to a modified method. Briefly, to remove non-specific inhibitors, 20 µL of serum were mixed with 180 µL of 25% kaolin and incubated for 30 min.
The extracted RNA was eluted in 50 µL of RNase- and DNase-free water. The RT-PCR assay was performed with specific primers (PIV5F1, PIV5R1, PIV5F2, PIV5R2, PIV5F3, and PIV5R3) that amplify the F gene of bPIV5 (Table 1). RT-PCR was carried out in a reaction mixture containing 10 µL of denatured RNA, 1 µL of each primer (50 pmol), 10 µL of 5× buffer (12.
The HI test is considered the standard method for detecting PIV5 antibodies [3]. Therefore, in this study, the HI test was used to screen for bPIV5 antibodies and confirm bPIV5 infection in Korean cattle.
These contributions are important because data on bPIV5 incidence can be used for vaccine development and initiating measures to block virus transmission. Therefore, in this study, we conducted a serological survey to determine the prevalence of antibodies against bPIV5 in cattle and optimize the reaction conditions for a bPIV5 RT-PCR assay.
성능/효과
In conclusion, based on the serosurveillance of bPIV5, a small number of Korean cattle have been exposed to bPIV5, which may cause respiratory disease under poor hygienic conditions. The RT-PCR assay described here was both sensitive and specific for the detection of bPIV5.
The results showed that the bPIVFR3 primer set was 10-fold more sensitive (2 × 102 TCID50/reaction) than the bPIVFR1 or bPIVFR2 primer set, and was able to amplify only the F gene of bPIV5 without any non-specific reaction.
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