1. DEVELOPMENT OF A NOVEL VNT ASSAY USING QRT-PCR-BASED ENDPOINT ASSESSMENT FOR RAPID DETECTION AND TITRATION OF NEUTRALIZING ANTIBODIES AGAINST FMDV Zhidong Zhang State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences(CAAS), China
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

2. Conclusion
A qRT-PCR-VNT was developed for rapid detection of neutralizing antibodies against FMDV Asia 1
The endpoint assessment was carried out as early as 20 hpi
The developed qRT-PCR-VNT with endpoint at 20hpi has good correlation with traditional VNT
Compared with traditional VNT, the developed qRT-PCR-VNT was robust with respect to assay duration (20 hours vs.72 hours)
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

3. Outline Background Results Conclusion
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

4. Outline Background Results Conclusion
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

5. Virus neutralization test
Diagnosis
Detection of FMDV neutralizing antibodies
Measure the serological response to FMDV infection and vaccination
Vaccine matching studies (in vitro)
Confirms clinical diagnosis
Supports the need for accurate clinical diagnosis
Full epidemiological information
Unknown/
Uncertainty
Known/
Certainty
Process
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

6. Virus neutralization test
Diagnosis
a confirmatory test
highly specific
But
Time-consuming
Slow, may take 2-3 days
Easily affected by man-made factors
Reading CPE
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

7. Real-time qRT-PCR assay
High sensitivity and specificity
Be quantitative
Results within hours
Widely used for detection of FMDV
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

8. qRT-PCR-based VNT assay
The qRT-PCR assay is used to quantify the reduction in FMDV replication resulting from sera neutralizing antibodies
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

9. qRT-PCR-based VNT assay
The RT-qPCR assay is used to quantify the reduction in FMDV replication resulting from sera neutralizing antibodies
FMDV
+ antiserum
Inoculation of IBRS 2 cells
RNA extraction
Quantification of viral RNA
Trizol added at 20 hpi
37oC， I h
37oC， 20 h
The mean % of reduction in FMDV RNA copy number
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

10. qRT-PCR-based VNT assay
The mean percentage of reduction in FMDV RNA copy number
= 1- (FMDV RNA copy number in serum sample /FMDV RNA copy number in non-neutralized virus control*) %
*non-neutralized virus control: cells infected with FMDV in absence of sera (virus control wells)
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

11. Outline Background Results Conclusion
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

12. Performance analysis of qRT-PCR assay targeting the 3D gene
Range spans 6 logs ranging from 2 to 8 log FMDV copies per reaction
Dilutions ranging from 1:10 to 1:107
Standard 3D RNA curve
RNA extracted from FMDV Asia-1/JSL/06)
Virus stock :105.6 TCID50/ 100μl
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

13. Assessment of FMDV replication kinetics by qRT-PCR
The replication kinetic of FMDV was determined by quantifying the RNA extracted from FMDV Asia 1-infected IBRS-2 cells grown in 96-well plates
100 TCID50/well of the virus
each point represents the mean value ± SD (n=5) of lg (copy number) against the hpi.
The mean of RNA copy number initially be of 1.3×103copieat 1 hour and increased to 107 copies at 20 h with absence of CPE.
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

14. Distribution of viral RNA copy for negative sera
Assessment of virus neutralization by qRT-PCR
Distribution of viral RNA copy for negative sera
negative sera collected from bovine (n=10), swine (n=5) and sheep (n=5)
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

15. Mean percentages of reduction
The mean percentages of reduction in FMDV RNA copy number in a manner dependent on the dilution of sera.
Dilution
Mean of RNA copy number SD
Mean percentages of reduction
1:4
3.19
0.96
63%
0.07
1:8
4.03
2.06
42%
0.19
1:16
6.46
2.02
24%
0. 12
1:32
7.69
0.36 8%
0.05
1:64
7.65
0.33 9%
1:128
7.77
control
8.36
0.16
23% cut off
Reduction of detectable FMDV copy number was observed in a manner dependent on the dilution of sera. The mean percentages of reduction regarding the negative sera were 63% (SD=0.07) at 1:4 dilution, 42% (SD=0.19) at 1:8 dilution, 24% (SD= 0. 05) at 1:16 dilution, 8% (SD=0.05) at 1:32 dilution, 9% (SD=0.05) at 1:64 dilution and 8% (SD=0.05) at 1:128 dilution. According to OIE manual[9], a sera neutralization titre of less than 1/16 is considered to be negative. The negative cut-off value was set by adding 3 standard deviations (SD) to the mean value of reduction in the FMDV copy number in comparison with a non-neutralized virus control (i.e. equal to or less than 23 % in reduction) at 1:32 dilution.
The negative cut-off value was set by adding 3 standard deviations (SD) to the mean value of reduction in the FMDV copy number in comparison with a non-neutralized virus control at 1:32 dilution
The negative： cut-off value =equal to or less than 23 % in reduction
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

16. Reduction of detectable FMDV copy number observed in sera collected from pigs infected with FMDV Asia 1 at 35 dpi
23% cut off
The mean percentages of the reduction at 1:32 is greater than 23 % in reduction
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

17. Analytical sensitivity
23% cut off
sera collected from pigs infected with FMDV Asia 1
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

18. Mean percentages of reduction
Reduction of detectable FMDV copy number observed in VNT-positive sera
Dilution
VNT
Mean of RNA copy number SD
Mean percentages of reduction
Sample 1 P
2.86
0.17
65.13%
Sample 2
2.93
0.32
64.27%
Sample 3
0.22
64.21%
Sample 4
2.79
0.10
65.95%
Sample 5
Sample 6
3.06
0.25
62.65%
Sample 7
3.03
0.26
63.07%
Sample 8
3.17
61.35%
Sample 9
2.82
0.30
65.61%
Control
N/A
8.20
The mean percentages of the reduction at 1:32 is greater than 23 % in reduction
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

19. 23% cut off
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

20. no cross-protection between different serotype of FMDV.
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

21. Correlation between Log2(VNT titre) and Log2(qRT-PCR-VNT titre)
Correlation between Log2(VNT titre) and Log2(qRT-PCR-VNT titre). 20 of FMDV negative sera were performed with VNT and qRT-PCR-VNT
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

22. CONCLUSION
A qRT-PCR-VNT was developed for rapid detection of neutralizing antibodies against FMDV Asia 1
The endpoint assessment was carried out as early as 20 hpi
The developed qRT-PCR-VNT with endpoint at 20hpi has good correlation with traditional VNT
Compared with traditional VNT, the developed qRT-PCR-VNT was robust with respect to assay duration (20 hours vs.72 hours)
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

23. Acknowledgements Yiming Song Yang Yang Xiangle Zhang Yanmin Li
Guozhen Cong
National Research Programe (2016YFD )
CAAS Innovation fund
CAAS master degree studentship
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

24. Thanks