Challenges for Providing Diagnostic Service: White Spot Disease (WSD)

Transcription

1 Regional Meeting of OIE Reference Centres in Asia and the Pacific6-7 February 2017, Tokyo, Japan Challenges for Providing Diagnostic Service: White Spot Disease (WSD) Grace Chu-Fang Lo National Cheng Kung University, Taiwan Reference Laboratory for White Spot Disease (Chinese Taipei) 1 February 6 th, 2017

2 Causative agent of WSD Taxonomy Family: Nimaviridae Genus: Whispovirus Type species: White spot syndrome virus WSSV genome A double-stranded circular DNA genome of over 300 kbp Over 90% of the WSSV ORFs show no significant similarity to other known proteins Uniqueness Very broad host range Replicates very rapidly in shrimp Lots of anti-host defense strategies A disease that is very difficult control Lo et al., 9 th ICTV report,

3 A losing battle for shrimp infected with WSSV When WSSV infects its crustacean host: The host ROS can be neutralized at initial stage of infection host defenses such as iron withholding defense mechanism, apoptosis and RNAi can be circumvented (e.g. by WSSVPK1, WSSV AAP1, WSSV RNAi suppressors) host defense proteins (e.g. NFkB and STAT) can even be co-opted by WSSV IE1 proteins the host can be overwhelmed by WSSV ICP11 protein

4 WSSV annexes shrimp STAT and NFkB to transactivate ie1 expression Liu et al., 2007; Huang et al., 2010; Wang et al., 2011

5 An explanatory model for the underlying STAT/ie1 mechanism in the outbreak of WSD in WSSV carrier shrimp under stressful conditions

6 Detection methods for WSSV carriers Best methods for carrier states are polymerase chain (PCR) methods Nested PCR (IQ2000 or equivalent ) and real-time PCR are recommended Ctrl. N P(10 3 )P(10 2 )P(10 1 ) Ctrl.: Shrimp internal control M: Marker N: Negative control P: Positive control ++++: Severe infection +++: Medium infection ++: Light infection +: Very light infection M 848 bp 580 bp 340 bp 6

7 Detection methods for diseased shrimp Antibody detection strips are best for diagnosing the patent disease state Shrimple, a commercially available antibody-based detection kit, is the most popular rapid test kit used to confirm the presence of the disease in shrimp Unfortunately, these cheap, easy to use kits are not sensitive enough to diagnose carrier shrimp, ie shrimp with a very low level of infection. 7

8 Pond-side diagnostic test for carrier shrimp Affordable Easy to use Accurate/Sensitive/Reliable

9 Comparison between PHINEX PCR and conventional PCR Time cost Machine price Sensitivity DNA preparation Power supply Data analysis Conventional PCR 1 2 hr 3,000 USD Lower Traditional 1hr. Stable V Computer PHINEX PCR 30 min 600 USD higher Simple 2 min Cellphone (developing) Cellphone (developing) Example of a shrimp pathogen detection (AHPND) PHINEX PCR Conventional PCR

10 PHINEX PCR developed by Prof Dar-Bin Shieh (NCKU) Compared to traditional PCR that heats and cools the sample from outside the PCR tube through a large heat block, PHINEX uses electromagnetic wave to pass the heat directly to reaction mixture. Overall diagnostic time is shortened by 50 %.

11 The outcome of WSSV carriers in a growout pond Pond contains WSSV-carrier shrimp Outcome of cultivation If we could develops an affordable easy to use and sensitive enough pond-side diagnostic kit, that would actually be quite useful for farmers: if farmers can easily and swiftly monitor whether or not the shrimp in a pond have become WSSV carrier, then they are in a position to modify their growout strategy accordingly

12 WSSV Transmission Pathways Another important factor in controlling WSD: How does the virus spread?

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14 Grading of shrimp fry and broodstock Infection status grading of shrimp fry Nauplii Nauplii HEAVY LIGHT NEGATIVE Nauplii : Heavily infected ( 50% of the tested batches were WSSV PCR-positive) Lightly infected ( 50% of the tested batches were WSSV PCR-positive) Negative (All of the tested batches were WSSV PCR-negative) 14

15 Prediction of offspring infection status WSSV infection in P. monodon brooders and their nauplii The offspring from heavily infected brooders are very likely to also be heavily infected by the virus. 15

16 Performance of WSSV shrimp seed in the field Performance of WSSV-infected and WSSVnegative P. monodon in grow out ponds 16

17 Hosts of WSSV WSSV infected cell WSSV Virus transmission is not just horizontal and vertical transmission from shrimp to shrimp but also both the environmental and a wide range of hosts 17

18 Copepod (Apocyclops royi) 18

19 18S rrna gene Continuous exposure to WSSV Sample collection: 0, 3, 24, 48, 72 hpi viral loads Genes expression 1st-step of PCR amplication

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21 Brooders immediately after capture S1 S2 S3 S N WSSV These brooders are from a hatchery in Vietnam They re in very good condition immediately after capture, no WSSV, everything is very clean. 21

22 Brooders from hatchery cultured for two days S5 S6 S N WSSV Two days later, we revisited the same hatchery and there was a problem Two of these shrimps are already showing a light infection with WSSV 22

23 Brooders from hatchery cultured for two weeks S8 S9 S N WSSV Following that initial light infection, two weeks later, all those of the tested shrimps were heavily infected 23

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25 C1C2C3C4C N WSSV Many hermit crabs were heavily infected with WSSV. Even thought they looked healthy, they had a very heavy virus load 25

26 Diagnosis, Quarantine and Disease Control Monitor and survey for its presence and then quarantine, or otherwise keep separate, the virus and its shrimp host. This is one of the benefits of the diagnostic services that we offer as an OIE reference lab.

27 From WSSV Emergence to Global Pandemic

28 Emergence of WSD in Emergence of WSD in ~ from East Asia WSD spread very quickly to become global pandemics 28

29 Look beyond Diagnosis and Quarantine During 2010 to 2012, WSSV caused severe mortalities in cultured penaeid shrimp in Saudi Arabia, Mozambique and Madagascar. In a 2013 study, Dr. Lightner s group suggested that the WSSV epidemics in these 3 countries were from a common source, and that this source was possibly the environment. It suggests that we need to look beyond diagnosis and quarantine. Right now, this is an enormous challenge.

30 Acknowledgements National Science Council, Ministry of Science and Technology Council of Agriculture, Executive Yuan National Cheng Kung University (NCKU) Prof. Lien I Hor Dr. Han Ching Wang Dr. I Tung Chen Dr. Shin Jen Lin Yun Tzu Huang National Taiwan University (NTU) Prof. Shin Shun Lin Graduate Institute of Translational Medicine, College of Medical Science and Technology, Taipei Medical University Dr. Hao Ching Wang

31 Thank you for your attention