1.
Open Session of the EuFMD - Cascais –Portugal 26-28 October 2016
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.
5
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
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences
Unknown/
Uncertainty
Known/
CertaintyProcess
Virus neutralization test

6.
6
• a confirmatory test
• highly specific
But
• Time-consuming
–Slow, may take 2-3 days
• Easily affected by man-made factors
–Reading CPE
Diagnosis
Virus neutralization test
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
FMDV
+ antiserum
37oC， I h 37oC， 20 h
Inoculation of
IBRS 2 cells
Trizol added
at 20 hpi
The mean % of
reduction in
FMDV RNA
copy number
Quantification
of viral RNA
RNA extraction
• The RT-qPCR assay is used to quantify the reduction in
FMDV replication resulting from sera neutralizing
antibodies
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
Standard 3D RNA curve RNA extracted from FMDV Asia-1/JSL/06)
• Dilutions ranging from 1:10 to 1:107
• Virus stock :105.6 TCID50/ 100μl
• Range spans 6 logs ranging from 2
to 8 log FMDV copies per reaction
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

13.
• 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.
Assessment of FMDV replication kinetics by qRT-PCR
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

14.
Assessment of virus neutralization by qRT-PCR
Distribution of viral RNA copy for negative sera
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

15.
OS16
• 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
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
percentage
s of
reduction
SD
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% 0.05
1:128 7.77 0.33 8% 0.05
control 8.36 0.16
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences
23% cut off

16.
OS16
The mean percentages of the reduction at 1:32 is greater than 23 % in reduction
Reduction of detectable FMDV copy number observed in sera collected from
pigs infected with FMDV Asia 1 at 35 dpi
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences
23% cut off

17.
OS16
Analytical sensitivity
sera collected from pigs infected with FMDV Asia 1
23% cut off
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

18.
OS16
The mean percentages of the reduction at 1:32 is greater than 23 % in 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 P 2.93 0.32 64.27%
Sample 3 P 2.93 0.22 64.21%
Sample 4 P 2.79 0.10 65.95%
Sample 5 P 2.79 0.10 65.95%
Sample 6 P 3.06 0.25 62.65%
Sample 7 P 3.03 0.26 63.07%
Sample 8 P 3.17 0.10 61.35%
Sample 9 P 2.82 0.30 65.61%
Control N/A 8.20 0.17
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

19.
OS16
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences
23% cut off

20.
OS16
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). 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.
• Yiming Song
• Yang Yang
• Xiangle Zhang
• Yanmin Li
• Guozhen Cong
• National Research Programe (2016YFD0500907)
• CAAS Innovation fund
• CAAS master degree studentship
Acknowledgements
Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

24.
Thanks